北部湾6个拟穴青蟹群体遗传多样性的ISSR分析

应用ISSR标记技术分析来自北部湾的拟穴青蟹6个地理群体(清化、党江、钦州湾、流沙湾、珍珠湾、闸口)的遗传变异和遗传结构,8条ISSR引物扩增111个个体,分析其中的66个位点,56个位点表现出多态性,表明拟穴青蟹在物种水平上的多态位点百分率是84.85％.拟穴青蟹6个群体的多态位点百分率为51.52％ ～63.64％,平均为57.58％.群体的遗传多样性按自高至低顺序排列为清化群体＞党江群体＞钦州湾群体＞流沙湾群体＞珍珠湾群体＞闸口群体.拟穴青蟹在种水平或群体水平上的多态位点百分率和Shannon信息指数表明,拟穴青蟹遗传多样性在甲壳类动物中处于较高层次,但比甲壳类以外的几类经济海产动物遗传多样性低.总的遗传分化系数GST值为0.1841,表明,总遗传变异的绝大部分存在于群体内,群体间的遗传分化程度较大.但是,绝大部分两两群体间的遗传分化系数值在0.0689 ～0.1276,为中等程度的遗传分化.AMOVA分析表明,群体内遗传变异占87.97％,群体间遗传变异占12.03％,群体间遗传分化程度中等但分化显著.Mantel test结果表明,拟穴青蟹6个群体间的遗传距离与地理距离之间的相关性不显著.聚类分析表明,来自广西沿海的4个群体聚成一支.     

Parentage assignment of the mud crab (Scylla paramamosain) based on microsatellite markers

In this study, microsatellite markers were employed to identify the parentage relationship in Scylla paramamosain. The exclusion probability of loci was found to be related with the level of their heterozygosity. When no parent information or only one parent information was available, the exclusion probability ranged from 22.0% to 56.6% and from 41.2% to 73.1%, with the combined exclusion probability for ten loci being 97.0% and 99.8%, respectively. The cumulative assignment success rate was 100% when no parent information was available using seven most informative microsatellite markers. Moreover, the power of the seven microsatellite markers for parentage assignment was tested by a double-blind test, which indicated that 95% of the progeny can be correctly assigned to their parents. This study provided a microsatellite-based approach for parentage assignment in S. paramamosain that will be useful for investigation of genetic background and molecular marker-assisted selective breeding in this important crab species.     

Erk2 in ovarian development of green mud crab Scylla paramamosain

We identified extracellular signal-regulated kinase 2 (erk2) from green mud crab, Scylla paramamosain, in this article. It was originally identified from an expressed sequence tag fragment from a normalized gonadal cDNA library. 5' Rapid amplification of cDNA end (RACE) technique was used to obtain the 5' untranslated region (UTR). The full-length cDNA of Sp-erk2 is 1516 bp, including a 5'-terminal UTR of 19 bp, an open-reading frame of 1098 bp, and a 3'-terminal UTR of 399 bp. The translated protein is 365 amino acids in length with a predicted molecular weight of 42 kDa, which is the same as other species. It is the first time that the expression of Sp-erk2 in different stages of ovary development of crustacean was analyzed, and the result showed that the expression of Sp-erk2 increased gradually with ovarian development, with a peak in the mature phase. In situ hybridization histochemistry was used to clarify the detail of expression. Positive signals illustrated that Sp-erk2 mRNA is present in follicular cells when the ovary is in early stages, and in both follicular cells and oocytes when it is in mature phases. All above suggest that Sp-erk2 is important for ovarian development.     

Reference gene and tropomyosin expression in mud crab Scylla olivacea,Scylla paramamosain and Scylla tranquebarica

Molecular Biology Reports - Tropomyosin, a muscle tissue protein is a major allergen in most of shellfish including mud crab. Quantitative real time-PCR (qRT-PCR) using a stable reference gene is...     

Molecular cloning and expression profiles of nitric oxide synthase (NOS) in mud crab Scylla paramamosain

The importance of the nitric oxide synthase (NOS) gene family is demonstrated by many studies in vertebrates and invertebrates in recent years. However, it keeps unknown of nitric oxide (NO) system and NOS gene family in mud crab Scylla paramamosain, an important cultured commercial crustacean in China and Pacific area. In this report, the cDNA of NOS containing full-length ORF was cloned from mud crab, S. paramamosain. It was of 4424 bp, including a 5′-terminal untranslated region (UTR) of 239 bp, a 3′-terminal UTR of 540 bp, which contained two ATTTA motifs, and an open reading frame (ORF) of 3645 bp encoding a polypeptide of 1214 amino acids. Structural analysis indicated that NOS contained a typical NO synthase domain at the N-terminal, next to a flavodoxin 1 domain, a flavin adenine dinucleotide (FAD) binding domain, respectively, and a conservative nicotinamide adenine dinucleotide (NAD) binding domain structure at the C-terminal. Quantitative real-time PCR analysis revealed S. paramamosain NOS (SpNOS) to be expressed in all tissues examined, with the highest expression in midintestine and the weakest level in heart and eyestalk. The expression profiles of SpNOS indicated that the NOS expression levels were significantly induced in midintestine, hepatopancrease and hemocytes after challenged with Vibrio Parahaemolyticus, the synthetic double-stranded RNA polyinosinic polycytidylic acid (poly I:C) and lipopolysaccharides (LPS). The NOS activity in hemocytes showed significant increase during at 24 h-48 h time period after immune challenges with V. Parahaemolyticus, poly I:C and LPS. Results here may suggest that the inducible NOS play an important role in mud crab’s defense against pathogenic infection.     

Comparative profiling of ovarian and testicular piRNAs in the mud crab Scylla paramamosain

PIWI-interacting RNAs (piRNAs) are abundantly found in germ cells and involved in gametogenesis and gonadal development. Information on the regulatory roles of piRNAs in crustacean reproduction, however, is scarce. Thus, we identified gonadal piRNAs of mud crab Scylla paramamosain. Of the 115,491 novel piRNAs, 596 were differentially expressed. Subsequently, 389,887 potential piRNA-target genes were predicted. The expression of 4 piRNAs and 9 genes with high piRNA interactions were validated with the inclusion of additional immature specimens, including LRP2 that is involved in growth and reproduction, MDN1 in ribosome biogenesis pathway and gametogenesis, and PRKDC, a DNA repair gene involved in gonadal differentiation and maturation. KEGG analysis further revealed the involvement of predicted piRNA target genes in gametogenesis- and reproduction-related pathways. Our findings provide baseline information of mud crab piRNAs and their differential expression between testes and ovaries suggests that piRNAs play an essential role in regulating gametogenesis and gonadal development.     

基于线粒体DNA的中国亚洲象种群遗传多样性及种群遗传结构

亚洲象是我国国家一级保护动物.本文利用非损伤性取样法,以亚洲象粪便中脱落的肠道上皮细胞为DNA来源,选用线粒体DNA作为分子标记,对分布于我国境内的亚洲象种群的遗传结构和种群遗传多样性进行研究.本研究得到mtDNA序列片段长度为556 bp,经对178个个体进行扩增结果分析,共得到24个单倍型.在5个地理种群中,除南滚河种群外,其他4个种群中的114个个体共享同一单倍型,南滚河种群与其他种群间未观察到共享单倍型.系统发生分析,观察到中国境内现有亚洲象种群在进化上分为两大分支,α和β.其中分支α中包含除南滚河种群外的4个地理种群,分支β仅含有南滚河种群,表明南滚河种群与其他4个地理种群间存在明显分化.遗传多样性分析结果表明,中国境内的亚洲象种群的遗传多样性水平较低,分析原因认为是栖息地破碎化阻断了种群间有效的基因交流.     

Rapid isolation and characterization of polymorphic microsatellite loci in the mud crab, Scylla paramamosain (Portunidae)

Scylla paramamosain is a widespread and commercially important species of coastal marine crab. We identified 13 polymorphic microsatellite loci from a genome library constructed with 5'-anchored PCR method. Thirty-two S. paramamosain from the East China Sea were used to analyze the characteristics of these loci. The number of alleles per locus ranged from 3 to 8, with a mean of 5.923. Observed and expected heterozygosities ranged from 0.500 to 0.875 and from 0.500 to 0.859, respectively. Eleven of the 13 loci were highly polymorphic (polymorphic information content >0.5). All of the 13 novel loci were in Hardy-Weinberg equilibrium after Bonferroni's correction (P < 0.0038). There was no null allele, stuttering errors or evidence of allelic dropout in any of the loci analyzed by MICRO-CHECKER. According to pairwise tests, no significant linkage disequilibrium was found among the 13 loci (P < 0.0038, adjusted value). These novel developed microsatellites will be useful for studies of genetic variation, population structure, conservation genetics, and molecular-assisted selective breeding of S. paramamosain.     

Inhibitory effects of the androgenic gland on ovarian development in the mud crab Scylla paramamosain

Isolation and characterization of androgenic hormone in decapod crustaceans depend on an effective bioassay of its action. In the present study, the effect of androgenic gland on ovarian development in the mud crab Scylla paramamosain was investigated with a view to develop a bioassay for androgenic hormone. Ovarian regression with degeneration of oocytes occurred in some female crabs implanted with androgenic gland in vivo. In vitro incubation of ovarian tissues at secondary vitellogenesis in extract of androgenic gland resulted in a significant decrease in amino acid uptake by the tissues. We propose that this inhibitory effect could be established as an effective bioassay for the isolation of androgenic hormone in the mud crab.     

Simultaneous estimation of mixing rates and genetic drift under successive sampling of genetic markers with application to the mud crab (Scylla paramamosain) in Japan

In stock enhancement programs, it is important to assess mixing rates of released individuals in stocks. For this purpose, genetic stock identification has been applied. The allele frequencies in a composite population are expressed as a mixture of the allele frequencies in the natural and released populations. The estimation of mixing rates is possible, under successive sampling from the composite population, on the basis of temporal changes in allele frequencies. The allele frequencies in the natural population may be estimated from those of the composite population in the preceding year. However, it should be noted that these frequencies can vary between generations due to genetic drift. In this article, we develop a new method for simultaneous estimation of mixing rates and genetic drift in a stock enhancement program. Numerical simulation shows that our procedure estimates the mixing rate with little bias. Although the genetic drift is underestimated when the amount of information is small, reduction of the bias is possible by analyzing multiple unlinked loci. The method was applied to real data on mud crab stocking, and the result showed a yearly variation in the mixing rate.     

Characterization of 40 single nucleotide polymorphism (SNP) via T m-shift assay in the mud crab (Scylla paramamosain)

In this study, single nucleotide polymorphism (SNP) were identified, confirmed and genotyped in the mud crab (Scylla paramamosain) using T _m-shift assay. High quality sequences (13, 311 bp long) were obtained by re-sequencing that contained 91 SNPs, with a density of one SNP every 146 bp. Of all 91 SNPs, 40 were successfully genotyped and characterized using 30 wild specimens by T _m-shift assay. The minor allele frequency per locus ranged from 0.017 to 0.500. The observed and expected heterozygosity, and polymorphism information content (PIC) ranged from 0.000 to 0.600, from 0.033 to 0.509, and from 0.033 to 0.375, respectively, with an average of 0.142, 0.239 and 0.198 per locus. Seventeen SNPs were significantly deviated from Hardy–Weinberg equilibrium. No significant linkage disequilibrium between pairs of loci was detected after sequential Bonferroni correction (P > 0.00125). Seventeen SNPs were related with known function genes. This study provided new molecular markers for investigation of population genetic diversity, construction of genetic linkage maps and molecular marker-assisted selection in this important crustacean species.     

Characterization and expression of calcium-activated potassium channels (Slo) in ovary of the mud crab,Scylla paramamosain

Large conductance calcium-activated potassium channels (Slo) play important roles in controlling neuronal excitability. At present, very little is known about the function of Slo channels on ovarian development. We cloned the SPSlo gene from the mud crab, Scylla paramamosain. This gene shows 91 and 93 % sequence identity to PISlo from the spiny lobster, Panulirus interruptus and CBSlo from the jonah crab, Cancer borealis, respectively. We isolated six variants of the SPSlo cDNA within S. paramamosain ovary tissue. Sequence analysis indicated that there were at least seven alternative sites in SPSlo, each with multiple alternative segments. Real-time PCR showed that the SPSlo gene was expressed in various tissues, and highly expressed in brain and ovary. In addition, the expression of SPSlo changed throughout ovarian development, highest at the early-developing stage (Stage II) followed by a slow decrease in subsequent stages. These results suggested that SPSlo channels may be implicated in the ovarian development of the mud crab.     

Changes in progesterone levels and distribution of progesterone receptor during vitellogenesis in the female mud crab (Scylla paramamosain)

Vertebrate-type steroids, such as progesterone, have been identified in crustaceans. The physiological activity of progesterone during vitellogenesis is still not well understood. In this study, progesterone levels in the female mud crab, Scylla paramamosain, were determined by enzyme-linked immunosorbent assay. Peak levels of progesterone were detected during the previtellogenic stage in the hemolymph, ovary, and hepatopancreas, whereas the progesterone level decreased significantly in vitellogenic stage I. During vitellogenic stage II, progesterone levels rose again in the hemolymph and ovary, but continued to decrease in the hepatopancreas. By using western blotting, progesterone receptor (PR), with an apparent molecular weight of 70 kDa, was identified in the ovary during both vitellogenic stages I and II. By means of immunohistochemistry, PR was detected mainly in the follicle cells during vitellogenic stage I and in the nuclei of oocytes in vitellogenic stage II. Our results strongly suggest that progesterone promotes vitellogenesis in the mud crab, S. paramamosain via a classical genomic mechanism.