Chinese Journal of Oceanology and Limnology   2017, Vol. 35 issue(2): 235-243     PDF       
http://dx.doi.org/10.1007/s00343-016-5288-6
Institute of Oceanology, Chinese Academy of Sciences
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Article Information

LIU Meng(刘盟), LIU Yuan(刘媛), HUI Min(惠敏), SONG Chengwen(宋呈文), CUI Zhaoxia(崔朝霞)
Polymorphisms of clip domain serine proteinase and serine proteinase homolog in the swimming crab Portunus trituberculatus and their association with Vibrio alginolyticus
Chinese Journal of Oceanology and Limnology, 35(2): 235-243
http://dx.doi.org/10.1007/s00343-016-5288-6

Article History

Received Nov. 19, 2015
accepted in principle Feb. 1, 2016
accepted for publication Feb. 25, 2016
Polymorphisms of clip domain serine proteinase and serine proteinase homolog in the swimming crab Portunus trituberculatus and their association with Vibrio alginolyticus
LIU Meng(刘盟)1,2, LIU Yuan(刘媛)2, HUI Min(惠敏)2, SONG Chengwen(宋呈文)2, CUI Zhaoxia(崔朝霞)2,3,4        
1 College of Fisheries and Life Science, Dalian Ocean University, Dalian 116023, China;
2 Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China;
3 Laboratory for Marine Biology and Biotechnology, Qingdao National Laboratory for Marine Science and Technology, Qingdao 266071, China;
4 National & Local Joint Engineering Laboratory of Ecological Mariculture, Qingdao 266071, China
ABSTRACT: Clip domain serine proteases (cSPs) and their homologs (SPHs) play an important role in various biological processes that are essential components of extracellular signaling cascades, especially in the innate immune responses of invertebrates. Here, polymorphisms of PtcSP and PtSPH from the swimming crab Portunus trituberculatus were investigated to explore their association with resistance/susceptibility to Vibrio alginolyticus. Polymorphic loci were identified using Clustal X, and characterized with SPSS 16.0 software, and then the significance of genotype and allele frequencies between resistant and susceptible stocks was determined by a χ2 test. A total of 109 and 77 single nucleotide polymorphisms (SNPs) were identified in the genomic fragments of PtcSP and PtSPH, respectively. Notably, nearly half of PtSPH polymorphisms were found in the non-coding exon 1. Fourteen SNPs investigated were significantly associated with susceptibility/resistance to V. alginolyticus (P < 0.05). Among them, eight SNPs were observed in introns, and one synonymous, four non-synonymous SNPs and one ins-del were found in coding exons. In addition, five simple sequence repeats (SSRs) were detected in intron 3 of PtcSP. Although there was no statistically significant difference of allele frequencies, the SSRs showed different polymorphic alleles on the basis of the repeat number between resistant and susceptible stocks. After further validation, polymorphisms investigated here might be applied to select potential molecular markers of P. trituberculatus with resistance to V. alginolyticus.
Key words: Portunus trituberculatus     clip domain serine proteinase     serine proteinase homolog     polymorphism     susceptibility/resistance    
1 INTRODUCTION

The swimming crab Portunus trituberculatus (Miers, 1876) supports a large crab fishery and aquaculture in China. However, with the development of intensive culture, various diseases caused by bacteria and viruses occur frequently in cultured P. trituberculatus stocks (Wan et al., 2011). The emulsification disease, which causes high mortality in P. trituberculatus, is mainly induced by infection with Vibrio alginolyticus (Wang et al., 2006). Therefore, breeding new strains of P. trituberculatus that are resistant to V. alginolyticus is considered an effective solution to control this disease.

Traditional selective breeding techniques are always expensive, time-consuming, and easily influenced by the environment, and they do not fulfill the urgent need for resistant strains. One of the methods that could be used to improve breeding strategies is marker-assisted selection (MAS), which is already successfully used in the improvement of agricultural populations (Dekkers and Hospital, 2002). Among all kinds of DNA markers, single nucleotide polymorphism (SNPs) have been the most widely used owing to a single base change in the DNA sequence and high genome density (Vignal et al., 2002).

Polymorphisms of the immune-related genes, which change their quality or quantity, could affect the immune capacity of individuals to protect themselves against infection (Li et al., 2009). In aquatic animals, these immunity-associated SNPs and simple sequence repeats (SSRs) have been discovered from many immune-related genes, such as the lysozyme gene in Zhikong scallop (Li et al., 2009), i-type lysozyme gene in the clam Meretrix meretrix (Yue et al., 2012), Hsp70 in Litopenaeus vannamei (Zeng et al., 2008), the MDA5 gene in grass carp (Wang et al., 2012), and the superoxide dismutase gene family in the bay scallop (Argopecten irradians) (Bao et al., 2010). As the essential components of extracellular signaling cascades, clip domain serine proteases (cSPs) and their proteolytically inactive homologs (SPHs) have been reported to play significant roles in crab innate immunity (Cui et al., 2010; Song et al., 2013a). However, the polymorphisms of cSP and SPH are rarely studied and the correlation between gene polymorphisms and disease susceptibility/resistance is still unknown.

In the present study, the genetic polymorphisms of PtcSP (Song et al., 2013a) and PtSPH (Cui et al., 2010) in P. trituberculatus are reported. In addition, the association between the polymorphisms and susceptibility/resistance of P. trituberculatus to V. alginolyticus is investigated. The aim of the research is to select some molecular markers for selective breeding of this crab species.

2 MATERIAL AND METHOD 2.1 Crab breeding and V. alginolyticus challenge

Two hundred P. trituberculatus were collected from a commercial farm (Qingdao, China) and fed with clam meat once every night, and their seawater was changed every day. During the entire period of the experiment, crabs were randomly divided into five groups (40 individuals in each group). Crabs from the bacteria-challenged groups received an injection of 100 μL live V. alginolyticus suspended in 0.1 mol/L phosphate buffer saline (PBS) (pH 7.0, 5×108 cfu/mL) at the arthrodial membrane of the last walking leg. A group of untreated crabs was used as a blank group, and control group crabs received an injection of 100 μL PBS. All crabs were observed every hour to identify dead ones until they were sampled after 130 h post-challenge. The dead crabs found in the early part of the study were regarded as susceptible stock, and the crabs that survived were identified as resistant stock. Muscles of all crabs were removed and kept at -20°C until genomic DNA was extracted.

2.2 Sampling, DNA extraction and primers

For SNP confirmation and SNP-loci association studies, appendages were obtained from the two stocks. Genomic DNA was extracted from the muscle of P. trituberculatus using a standard phenolchloroform extraction method. Two pairs of genespecific primers (cSP-F and cSP-R, and SPH-F and SPH-R; see Table 1) were designed based on the obtained DNA sequences (GenBank accession nos. JF412651 for PtcSP and FJ769222 for PtSPH) to acquire the fragments of genomic DNA from resistant and susceptible stocks.

Table 1 Sequences of the primers used in this study
2.3 PCR amplification and sequencing

PCR amplification was carried out in a 25-μL reaction volume containing 2.5 μL of 10× PCR buffer (TransGen), 0.5 μL of dNTP (10 mmol/L), 0.5 μL each of forward primer and reverse primer (10 mmol/L), 0.2 μL (1 U) of EasyTaq DNA polymerase (TransGen), 1 μL of template DNA, and 19.8 μL of sterile distilled H2O. The reaction was performed in a TaKaRa PCR Thermal Cycler Dice Model TP600 (TaKaRa Bio Inc.) with the following conditions: one cycle at 94°C for 3 min, followed by 34 cycles of 94°C for 30 s, 50°C for 50 s and 72°C for 2 min, and a final 10-min extension at 72°C.

The PCR products from 30 susceptible crabs and 30 resistant crabs were detected by electrophoresis on 1% agarose gels and purified by the manufacturer’s protocol of a PCR gel purification kit (Axygen), and then the objective fragments were ligated with pMD19-T simple vector (TaKaRa, Dalian, China). The vector was transformed into Escherichia coli Trans1-T1 (TransGen, Beijing, China). At least three positive recombinant clones were identified through anti-Amp selection and the PCR products were sequenced with M13 primers (Table 1) using an ABI3730 Automated Sequencer (Applied Biosystems).

2.4 Identification and analysis of SNPs in PtcSP and PtSPH genes

Polymorphisms of PtcSP and PtSPH genes were identified and analyzed using Clustal X. Statistical analysis of SNPs was carried out with SPSS 16.0 software. Significant difference of genotype and allele frequencies between the two stocks was determined by χ2. P values less than 0.05 and 0.01 were considered statistically significant.

2.5 Identification and analysis of SSRs in the PtcSP gene

The allele (or haplotype) of SSR from each sample was identified and the repeat number of each unit was counted with the software SSRHunter. A chi-squared test was applied to analyze the difference of repeat number frequencies and identify the association with V. alginolyticus between resistant and susceptible stocks.

3 RESULT 3.1 Identification of susceptible and resistant crabs

Among 120 infected crabs, we found the first dead individual 7 h after challenging with V. alginolyticus. The 30 crabs that died before 100 h were regarded as being relatively susceptible to V. alginolyticus (susceptible stock), whereas 32 that survived 130 h post-challenge were classified as being relatively resistant to V. alginolyticus (resistant stock). Few dead crabs were found in blank and control groups.

3.2 Analysis of SNPs in the PtcSP gene 3.2.1 SNPs of PtcSP gene and association with V. alginolyticus-resistance

A 2 355-bp fragment of PtcSP gene was obtained. By direct sequencing from 22 susceptible specimens and 22 resistant specimens of P. trituberculatus, 109 SNPs including 77 transitions, 22 transversions and 10 ins-dels were detected (Table 2 and Fig. 1a). Of these SNPs, 66 were observed in introns, 42 in coding exons and one in non-coding exons. In the coding region, 16 synonymous mutations and 26 nonsynonymous mutations were found in PtcSP (Table 2 and Fig. 1b). The ratio of non-synonymous/ synonymous (dN/dS) was 1.6, which is widely used as an indicator of selective pressure, suggesting that the PtcSP gene had evolved under positive selection (dN/dS>1) (Chen and Sun, 2011). The deletion of C799, G1480 and CG(1750-1751) led to the early termination of the open reading frame (ORF).

Table 2 Statistics of SNPs in PtcSP
Figure 1 Polymorphisms of PtcSP in P. trituberculatus a. polymorphisms of PtcSP genomic DNA fragment. The nucleotides are numbered on the right. Sequences of introns are shadowed. Start and stop codons are boxed. SNP loci are underlined and the variants are described below; b. deduced amino acids of PtcSP. Synonymous mutations are marked with arrowheads. Non-synonymous mutations are underlined and the variations are described below. Early termination mutation is marked with an asterisk.

Genotype and allele frequencies of SNPs in PtcSP were numbered and analyzed between susceptible and resistant stocks (Table 3). Eleven polymorphic sites were found to be significantly different in the two stocks by examining the distributions of polymorphisms with χ2 tests, suggesting that they might be associated with susceptibility/resistance to V. alginolyticus. One SNP (I1-(170-175) AACAGTIns/del) exhibited a significant difference in genotype frequencies (P < 0.05) and an extremely significant difference in allele frequencies (P < 0.01) in the two stocks, of which the AACAGT-Ins allele was more prevalent in the resistant stock (59.1%) than the susceptible stock (22.7%). Another 10 SNPs were significantly different in allele frequencies (P < 0.05) between susceptible and resistant stocks.

Table 3 Distribution of PtcSP SNPs in susceptible and resistant stocks
3.2.2 SSRs in the PtcSP gene

Five SSRs, (AGG)n, (AGG)nAA (GAG)m, (AGG)nTA (GAG)m, (AGG)nTG (GAG)m and (AGG)nTGGTG (GAG)m, were detected in intron 3 of the PtcSP gene. No statistically significant difference in polymorphisms of these SSRs was observed between susceptible and resistant stocks (Table 4). However, some differences were found by analysis of the statistics. Among the five alleles of the unit based on the difference of repeat number of each unit, (AGG)10 demonstrated the highest frequency (66.7% in susceptible crabs and 83.3% in resistant crabs), followed by (AGG)6-9 (13.3% and 16.7%, respectively). Similarly, for the polymorphism of (AGG)4TGGTG (GAG)5, only two crabs were detected in resistant crabs and the others were not found.

Table 4 Distribution of PtcSP SSRs in susceptible and resistant stocks
3.3 Analysis of SNPs in PtSPH gene

The genomic sequence of PtSPH was amplified from 21 susceptible samples and 21 resistant samples. In the 1965-bp fragment, 77 SNP polymorphisms were detected, including 53 transitions, 18 transversions and six ins-dels (Table 5 and Fig. 2a). Of these, 20 polymorphic sites were detected in introns, 19 in coding exons and 38 in non-coding exons. Among 19 SNPs in the coding region, 12 synonymous mutations and seven non-synonymous mutations were observed in the PtSPH (Table 5 and Fig. 2b). The non-synonymous/synonymous ratio (dN/dS=0.58) was lower than the expected ratio (ratio=1) under neutral evolution, which may result from some sites of the PtSPH gene being under negative (purifying) selection for functional constraints.

Table 5 Statistics of SNPs in PtSPH
Figure 2 Polymorphisms of PtSPH in P. trituberculatus a. polymorphisms of PtSPH genomic DNA fragment. The nucleotides are numbered on the right. Sequences of introns are shadowed. Start and stop codons are boxed. SNP loci are underlined and the variants are described below; b. deduced amino acids of PtSPH. Synonymous mutations are marked with arrowheads. Non-synonymous mutations are underlined and the variations are described below.

Genotype and allele frequencies of SNPs in PtSPH were analyzed and confirmed between susceptible and resistant stocks with the software SPSS 16.0 (Table 6). According to the χ2 test, three SNP loci (I1-326, I1-524 and I2-607) showed significant differences only in allele frequencies (P < 0.05).

Table 6 Distribution of PtSPH SNPs in susceptible and resistant stocks
4 DISCUSSION

A variety of diseases, especially those caused by V. alginolyticus, are causing serious problems for the cultivation of swimming crabs in China (Liu et al., 2007). The selective culture of P. trituberculatus by traditional methods in our laboratory has revealed that some were disease resistant. Hence, it is crucial to understand their molecular immunity to enhance resistance of cultured swimming crabs to diseases. In this study, PtcSP and PtSPH were selected to be candidate genes for polymorphic identification, which were previously found in our lab, and the association analysis with resistance to V. alginolyticus was also investigated.

Based on the genomic structure, the polymorphisms in exons and adjacent introns of PtcSP and PtSPH were further analyzed. A total of 186 SNPs including 86 in introns, 39 in non-coding exons, and 61 in coding exons were detected. Our identified SNPs were found more often in non-coding regions than in coding regions, which may be because of a relatively high conservation of the coding region sequence (Liu, 2007). It is worth noting that 34 SNPs accounting for 44.2% of PtSPH variations were found in non-coding exon 1. To our knowledge, this is the first report on polymorphisms of the non-coding region before the initiator ATG codon in P. trituberculatus. Further investigations on the genetic polymorphisms of longer upstream regulatory sequences may be helpful to find more potential V. alginolyticus-resistanceassociated markers in PtcSP and PtSPH.

In our study, the most detected SNPs were not significantly different between susceptible and resistant stocks. Fourteen SNPs were found to be correlated with the resistance of P. trituberculatus to V. alginolyticus. Of these, eight intronic SNPs, especially locus I1-(170-175) in PtcSP, were associated significantly with the Vibrio susceptibility/ resistance trait. These trait-related intronic SNPs are also reported in the crustacean hyperglycemic hormone gene of Macrobrachium rosenbergii (Thanh et al., 2010) and i-type lysozyme gene of Meretrix meretrix (Yue et al., 2012). Mutations in the introns do not directly participate in the process of translation; however, they may cause splicing abnormalities and affect gene expression (Pagani and Baralle, 2004).

Among SNPs associated with V. alginolyticusresistance in coding exons, only one synonymous SNP (E5-1457 T-C) in PtcSP was found. The synonymous SNP is also detected in PtALFs of P. trituberculatus (Li et al., 2013) and human DRD2 gene (Duan et al., 2003). Though synonymous mutations do not impact biological traits directly, they are indispensable for their important roles in regulations or modifications of gene expression and protein processing (Hershberg and Petrov, 2008). In addition, four non-synonymous SNPs, E1-38 A-G, E1-53 A-G, E1-63 C-G and E8-2289 G-A, and one ins-del (E5-1480 G) in PtcSP with resistance to V. alginolyticus were identified. A similar result also occurs in C-type lectin (Hao et al., 2015) and Crustin genes (Song et al., 2013b) of P. trituberculatus. Such non-synonymous and ins-del variations may cause considerable differences in the composition and structure of protein, and potentially affect the immunity phenotypes of crabs. All these polymorphic loci could be candidate gene markers with enhanced resistance to V. alginolyticus and might be applied in further selective breeding of P. trituberculatus.

Microsatellites are widely used DNA markers because of their abundance, small locus size, reproducibility, and high levels of polymorphism (Liu and Cordes, 2004). In our study, some SSRs were found only in PtcSP. Consistent with the report that distributions of SSRs in coding regions are relatively rarer than in non-coding regions (Hancock, 1995), these identified SSRs are all in introns of PtcSP. They may play important roles in population structure, genetic diversity, and the association analysis with resistance. Unlike the report of PtCrustin2 gene in Song et al. (2013b), the detected SSRs of PtcSP showed no statistically significant association between SSRs and resistance/susceptibility to V. alginolyticus. However, some differences were found when comparing the repeat number of these SSRs between susceptible and resistant stocks, which is also observed in PtALFs of P. trituberculatus (Li et al., 2013). Therefore, it is still unknown whether the identified SSRs in swimming crab can be regarded as potential V. alginolyticus-resistance markers.

5 CONCLUSION

We investigated the distribution of polymorphisms in PtcSP and PtSPH and the association with the resistance of swimming crab to V. alginolyticus. A total of 14 SNPs, especially the genotype and allele of I1-(170-175) AACAGT-Ins/del site in the PtcSP, can be considered V. alginolyticus-resistance candidate molecular markers in P. trituberculatus, which could potentially be applied in the future selective breeding of resistant crabs and controlling diseases caused by Gram-negative bacteria.

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