Genetic linkage map of bay scallop, Argopecten irradians irradians (Lamarck 1819)

The bay scallop (Argopecten irradians irradians Lamarck 1819) has become one of the most important aquaculture species in China. Genetic improvement of cultured bay scallop can benefit greatly from a better understanding of its genome. In this study, we developed amplified fragment length polymorphisms (AFLPs) and simple sequence repeat markers from expressed sequence tags (EST-SSRs) for linkage analysis in bay scallop. Segregation of 390 AFLP and eight SSR markers was analysed in a mapping population of 97 progeny. Of the AFLP markers analysed, 326 segregated in the expected 1:1 Mendelian ratio, while the remaining 74 (or 19.0%) showed significant deviation, with 33 (44.6%) being deficient in heterozygotes (A/a). Among the eight polymorphic EST-SSR loci, one marker (12.5%) was found skewing from its expected Mendelian ratios. Eighteen per cent of the markers segregating from female parent were distorted compared with 21% of the markers segregating from male parent. The female map included 147 markers in 17 linkage groups (LGs) and covered 1892.4 cM of the genome. In the male map, totally 146 AFLP and SSR markers were grouped in 18 LGs spanning 1937.1 cM. The average inter-marker spacing in female and male map was 12.9 and 13.3 cM respectively. The AFLP and SSR markers were distributed evenly throughout the genome except for a few large gaps over 20 cM. Although preliminary, the genetic maps presented here provide a starting point for the mapping of the bay scallop genome.

Molecular cloning and mRNA expression of peptidoglycan recognition protein (PGRP) gene in bay scallop (Argopecten irradians, Lamarck 1819)

Peptidoglycan recognition proteins (PGRPs) are a type of pattern recognition molecules (PRM) that recognize the unique cell wall component peptidoglycan (PGN) of bacteria and are involved in innate immunity. The first bivalve PGRP cDNA sequence was cloned from bay scallop Argopecten irradians by expressed sequence tag (EST) and PCR technique. The full-length cDNA of bay scallop PGRP (designated AiPGRP) gene contained 10 18 bp with a 615-bp open reading frame that encoded a polypeptide of 205 amino acids. The predicted amino acid sequence of AiPGRP shared high identity with PGRP in other organisms, such as PGRP precursor in Trichoplusia ni and PGRP SC2 in Drosophila melanogaster. A quantitative reverse transcriptase Real-Time PCR (qRT-PCR) assay was developed to assess the mRNA expression of AiPGRP in different tissues and the temporal expression of AiPGRP in the mixed primary cultured hemocytes challenged by microbial components lipopolyssacharide (LPS) from Escherichia coli and PGN from Micrococcus luteus. Higher-level mRNA expression of AiPGRP was detected in the tissues of hemocytes, gonad and kidney. The expression of AiPGRP in the mixed primary cultured hemocytes was up regulated after stimulated by PGN, while LPS from E. coli did not induce AiPGRP expression. The results indicated that AiPGRP was a constitutive and inducible expressed protein that was mainly induced by PGN and could be involved in scallop immune response against Gram-positive bacteria infection. (c) 2006 Elsevier Ltd. All rights reserved.

The cDNA cloning and mRNA expression of heat shock protein 70 gene in the haemocytes of bay scallop (Argopecten irradians, Lamarck 1819) responding to bacteria challenge and naphthalin stress

Heat shock protein 70 (HSP70) is an important member of the heat shock protein superfamily, and it plays a key role in the process of protecting cells, facilitating the folding of nascent peptides and responding to stress. The cDNA of bay scallop Argopecten irradians HSP70 (designated AIHSP70) was cloned by the techniques of homological cloning and rapid amplification of cDNA end (RACE). The full length of AIHSP70 cDNA was 2651 bp in length, having a 5' untranslated region (UTR) of 96 bp, a 3' UTR of 575 bp, and an open reading frame (ORF) of 1980 bp encoding a polypeptide of 659 amino acids with an estimated molecular mass of 71.80 kDa and an estimated isoelectric point of 5.26. BLAST analysis revealed that the AIHSP70 gene shared high identity with other known HSP70 genes. Three classical HSP signature motifs were detected in AIHSP70 by InterPro, analysis. 3-D structural prediction of AIHSP70 showed that its N terminal ATPase activity domain and,C terminal substrate-binding domain shared high similarity with that in human heat shock protein 70. The results indicated that the AIHSP70 was a member of the heat shock protein 70 family. A semi-quantitive RT-PCR method was used to analyse the expression of AIHSP70 gene after the treatment of naphthalin which is one kind of polycyclic aromatic hydrocarbon (PAH) and the challenge of bacteria. mRNA expression of AIHSP70 in scallop was up-regulated significantly after the stimulation of naphthalin and increased with increasing naphthalin concentration. A clearly time-dependent expression pattern of AIHSP70 was observed after the scallops were infected by Vibrio anguillarum, and the mRNA expression reached a maximum level at 8 h and lasted to 16 h, and then dropped progressively. The results indicated that AIHSP70 could play an important role in mediating the environmental stress and immune response in scallop. (c) 2006 Elsevier Ltd. All rights reserved.

Molecular cloning, characterization and expression of a novel serine proteinase inhibitor gene in bay scallops (Argopecten irradians, Lamarck 1819)

Serine protease inhibitors, critical regulators of endogenous proteases, are found in all multicellular organisms and play crucial roles in host physiological and immunological effector mechanisms. The first mollusk serine proteinase inhibitor (designated AISPI) cDNA was obtained from the bay scallop Argopecten irradians by randomly sequencing a whole tissue cDNA library and rapid amplification of cDNA ends (RACE). The full-length cDNA of the scallop serine protease inhibitor was 1020 bp, consisting of a 5'-terminal untranslated region (UTR) of 39 bp, a 3'-terminal UTR of 147 bp with a canonical polyadenylation signal sequence AATAAA and a poly(A) tail, and an open reading frame of 834 bp. The AISPI cDNA encoded a polypeptide of 278 amino acids with a putative signal peptide of 22 amino acids and a mature protein of 256 amino acids. The deduced amino-acid sequence of AISPI contained six tandem and homologous domains similar to that of Kazal-type serine protease inhibitors, including the conserved sequence C-X(7)-C-X(6)-Y-X(3)-C-X(2,3)-C and six cysteine residues responsible for the formation of disulfide bridges, indicating that the AISPI protein from bay scallop should be a member of the Kazal-type serine protease inhibitor family. The temporal expression of AISPI was measured by semi-quantitative RT-PCR after injury or bacterial challenge. After the adductor muscle was wounded or injected with Vibrio anguillarum, the expression of AISPI mRNA in hemolymph was up-regulated and reached the maximum level at 8 and 16 h, respectively, and then progressively dropped back to the original level. The results indicated that AISPI could play an important role in injury healing and immune response in mollusks as it could be induced by injury and bacterial challenge. (c) 2005 Elsevier Ltd. All rights reserved.

Effect of parental stock size on F-1 genetic structure in the bay scallop, Argopecten irradians (Lamarck, 1819)

Three F-1 families of the bay scallop, Argopecten irradians, were produced from one, two and 10 individuals. The genetic changes in these populations, which suffered recent and different levels of bottleneck, were analysed using amplified fragment length polymorphism (AFLP) techniques. In the parental stock, a total of 330 bands were detected using seven AFLP primer pairs, and 70% of the loci were polymorphic. All F-1 groups had a significantly lower proportion of polymorphic loci when compared with the initial stock, and loss of the rare loci and reduction in heterozygosity both occurred. The progeny of the larger population (i.e., N=10) exhibited a lesser amount of genetic differentiation compared with the progeny from N=2, which showed lesser differentiation than progeny from N=1. The effective population sizes (N-e) in N=1, 2 and 10 were estimated as 1.50, 1.61 and 2.49. Based on regression analysis, we recommend that at least 340 individuals be used in hatchery populations to maintain genetic variation.

Production of a base population and its responses to F-1 selection in the bay scallop, Argopecten irradians irradians Lamarck (1819)

A base population of the bay scallop, Argopecten irradians irradians Lamarck, was produced by crossing two cultured bay scallop populations. After 1 year of rearing, the top 10% truncation selection of the top 10% (i=1.755) was carried out in the base population of about 1300 adults. A control parental group with a an identical number to the select parental group was randomly selected from the entire population before isolation of the select parental group. The result showed that, at the larval stage, the growth rate of larvae in the selected line was significantly higher than that of the control (P < 0.05), and that the genetic gain was 6.78%. Owing to the lower density of control at the spat stage, the mean shell length of the control line was larger than that of the select line at day 100. When the same density was adjusted between two lines in the grow-out stage (from day 100 to 160), the daily growth rate of the selected line was significantly higher than that of the control line (P < 0.05). Survival of the select line was significantly larger than that of the control line in the grow-out stage. In conclusion, the results obtained from this experiment indicate that selective breeding from a base population with a high genetic diversity established by mass spawning between different populations appears to be a promising method of genetic improvement in bay scallop, A. irradians irradians Lamarck.

Different responses to selection in two stocks of the bay scallop, Argopecten irradians irradians Lamarck (1819)

Two different stocks (A and B) of the bay scallop Argopecten irradialls irradians (Lamarck, 1819) were used to test mass selection on growth. Stock A was a descending stock from the initial introduction from U.S.A. in 1982, which had been cultured in China for about 20 years. Stock B was the third generation from a recent introduction from U.S.A. in 1999. Truncation selection was conducted by selecting the largest 11% scallops in shell length from Stock A and the largest 12.7% scallops from Stock B as parents for the respective selected groups. Before the removal of parents for truncation selection, equal numbers of scallops were randomly chosen from Stock A and B to serve as parents for the control groups. Offspring from the four groups were reared under the same hatchery, nursery, and grow-out conditions. Values of response to selection and realized heritability at larvae, spat and grow-out stages for Stock B were all significantly (P < 0.001) higher than its counterpart for Stock A. For Stock A, no significant response to selection was observed (P > 0.05) at any stage, and the realized heritability for shell length was 0.015 +/- 0.024 for larvae, 0.040 +/- 0.027 for spat, and 0.080 +/- 0.009 for grow-out, respectively. For Stock B, however, significant (P < 0.05) response to selection was observed, and the realized heritability for shell length was 0.511 +/- 0.010 for larvae, 0.341 +/- 0.022 for spat, and 0.338 +/- 0.015 for grow-out. On average, responses to selection at the three stages for Stock B was 30 x, 7.1 x, and 3 x higher than its counterpart for Stock A, respectively. Accordingly, realized heritability at above stages for Stock B was 33 X, 7.5 x, and 3.2 X higher than its counterpart for Stock A, respectively. Differences in response to selection and realized heritability between the two stocks are presumably due to differences in genetic variability. As the 20th generation from the initial introduction consisted of only 26 scallops, Stock A is known to be highly inbred, while inbreeding in Stock B is negligible. (C) 2004 Elsevier B.V. All rights reserved.

Identification and mapping of amplified fragment length polymorphism markers linked to shell color in bay scallop, Argopecten irradians irradians (Lamarck, 1819)

Amplified fragment length polymorphisms (AFLP) were used to study the inheritance of shell color in Argopecten irradians. Two scallops, one with orange and the other with white shells, were used as parents to produce four F-1 families by selfing and outcrossing. Eighty-eight progeny, 37 orange and 51 white, were randomly selected from one of the families for segregation and mapping analysis with AFLP and microsatellite markers. Twenty-five AFLP primer pairs were screened, yielding 1138 fragments, among which 148 (13.0%) were polymorphic in two parents and segregated in progeny. Six AFLP markers showed significant (P < 0.05) association with shell color. All six loci were mapped to one linkage group. One of the markers, F1f335, is completely linked to the gene for orange shell, which we designated as Orange1, without any recombination in the progeny we sampled. The marker was amplified in the orange parent and all orange progeny, but absent in the white parent and all the white progeny. The close linkage between F1f335 and Orange1 was validated using bulk segregation analysis in two natural populations, and all our data indicate that F1f335 is specific for the shell color gene, Orange1. The genomic mapping of a shell color gene in bay scallop improves our understanding of shell color inheritance and may contribute to the breeding of molluscs with desired shell colors.

Inbreeding depression for various traits in two cultured populations of the American bay scallop, Argopecten irradians irradians Lamarck (1819) introduced into China

This paper examines the effect of inbreeding level of population on the magnitude of inbreeding depression expressed by comparing them between two cultured populations (A and B) in the hermaphroditic animal of the bay scallop Argopecten irradians irradians. Population A is expected to have less genetic variations and higher inbreeding level due to longer cultured history (20 generations) and less "ancestral" individuals (26 individuals) than population B due to shorter cultured history (4 generations) and more "ancestral" individuals (406 individuals). Two groups within each population were produced, one using self-fertilization and one using mass-mating within the same population. Selfed offspring (AS and BS) from two populations both had lower fitness components than their mass-mated counterparts (AM and BM) and exhibited inbreeding depression for all examined traits, e.g. lower hatching, less viability and slower growth, indicating that inbreeding depression is a common feature in this animal. Fitness components in all traits of offspring from population A significantly differed those from population B and the magnitude of inbreeding depression for all traits in population A with higher inbreeding level was significantly smaller than that in population B with lower inbreeding level, indicating that both fitness components and magnitude of inbreeding depression were significantly affected by inbreeding level of populations and genetic load harbored in population A may be partially purged through inbreeding. Moreover, the magnitude of inbreeding depression in the two populations both varied among traits and life history stages. The present results support the partial-dominance hypothesis of inbreeding depression. (C) 2008 Elsevier B.V. All rights reserved.

Sustained response to selection in an introduced population of the hermaphroditic bay scallop Argopecten irradians irradians Lamarck (1819)

Introduced species often start with limited genetic variability, which is problematic for selective breeding. The problem of inbreeding can be exasperated by hermaphroditism. The bay scallop Argopecten irradians irradians is a hermaphroditic species that has been introduced to and now supports a major aquaculture industry in China. Positive response to selection for fast growth was observed in one of the less inbred stocks in a previous study. In this study, we evaluated selection for the second generation to determine if response to selection can be sustained in this introduced population of a hermaphroditic species. Response to selection, realized heritability for the second generation, cumulative (over two generations), current (for the second generation) and residual (from the first generation) genetic gains were estimated by comparing three different types of lines: SS (selected for two generation), SC (selected for the first generation only) and CC (unselected for two generations). The SS line grew significantly faster (P < 0.05) than the other two lines, indicating that the second generation selection for faster growth is still effective. Response to selection and realized heritability for the second generation were 0.612 +/- 0.101 and 0.349 +/- 0.057, respectively, which are similar to those observed for the first generation. The cumulative, current, and residual gains were 17.56 +/- 5.30%, 10.63 +/- 2.46%, and 6.25 +/- 3.13%, respectively. The sustained response to selection for the second generation observed here suggests that considerable genetic variability exists in this population and that future efforts on selective breeding are likely to be fruitful. (c) 2005 Elsevier B.V. All rights reserved.