Xiande Liu

Microsatellite-centromere mapping in large yellow croaker (Pseudosciaena crocea) using gynogenetic diploid families.

The large yellow croaker (Pseudosciaena crocea) is an economically important marine fish in China. Inheritance of 22 heterozygous microsatellite loci was examined in normal crossed diploid families and meio-gynogenetic families in P. crocea. Two gynogenetic families were produced via inhibition of the second polar body in eggs fertilized with UV-irradiated sperm. The ratio of gynogenesis was proven to be 100% and 96.9% in the two families, respectively. Of the 22 examined loci, 4 showed a segregation distortion in both control and gynogenetic families. Microsatellite–centromere (M–C) map distances were examined using 18 loci with normal Mendelian segregation. Estimated recombination rates ranged between 0 and 1.0 under the assumption of complete interference. High recombinant frequencies between heterozygous markers and the centromere were found in large yellow croaker, as in other teleosts. The average recombination frequency was 0.586. Ten loci showed high M–C recombination with frequency greater than 0.67. M–C distances provide useful information for gene mapping in large yellow croaker.

Estimated genetic parameters for growth-related traits in large yellow croaker Larimichthys crocea using microsatellites to assign parentage

To estimate the heritabilities of growth-related traits in large yellow croaker, Larimichthys crocea, three independent full-factorial crosses were created by crossing four males × four females, seven males × three females and four males × three females (set 1, set 2 and set 3). At 13 months post-hatch, the juveniles were collected from three cross sets and measured for body mass (M), standard length (L(S)) and body height (H(B)). In addition, the parentage of the juveniles was assigned by genotyping six or seven polymorphic microsatellite loci. Out of the 959 juveniles, 99·6% could be assigned to a single parental pair. Heritabilities of growth-related traits were estimated for individual and combined sets with the pedigrees reconstructed by using microsatellite genotypes. The heritability estimates at 13 month-old were 0·02-0·30 for M, 0·02-0·25 for L(S) and 0·03-0·36 for H(B). These results showed that the heritabilities of M, L(S) and H(B) were different among three sets, which suggested that different breeding strategies should be adopted for different sets.

The cross breeding and genetic analysis of hybrids of Larimichthys crocea(♀)and Nibea miichthioides(♂)

To understand the feasibility of cross breeding between Larimichthys crocea ♀ and Nibea miichthioides ♂,two hybrid families were built(LN1 and LN2).Ploidy of F1 hybrids was identified.Moreover,ten microsatellite loci were used for genetic relationship analysis between F1 hybrids and their parents.The result showed that L.crocea(♀)and N.miichthioides(♂)can be successfully crossed and hybrid offspring of normal survival were produced.Survival rate of hybrid offspring at the age of 45 days post hatch reached about 30%.However,fertilization rate(29.0%,32.6%)and hatching rate(75.0%,76.7%)of two hybrid families were significantly lower than those in large yellow croaker families(P0.05).Appearance of juvenile hybrid fish is visibly different from those of large yellow croaker,which has characteristics of both parents:slender body,sharp blunt head and their body sides are covered with black-brown spots.The results of DNA relative content investigation and genetic relationship analysis demonstrated that more than 90% of the hybrid offspring were hybrid diploid,in addition,a small number of those were hybrid triploid and gynogenesis diploid.Till the age of 45 days,growth rate of both hybrid diploid and hybrid triploid was slower than that of large yellow croaker.These findings will provide reference for exploitation and management of F1 hybrids of L.crocea(♀)and N.miichthioides(♂).

De novo characterization of Larimichthys crocea transcrip-tome for growth-/immune-related gene identification and massive microsatellite (SSR) marker development

The large yellow croaker, Larimichthys crocea is an important marine fish in China with a high economic value. In the last decade, the stock conservation and aquaculture industry of this species have been facing severe challenges because of wild population collapse and degeneration of important economic traits. However, genes contributing to growth and immunity in L. crocea have not been thoroughly analyzed, and available molecular markers are still not sufficient for genetic resource management and molecular selection. In this work, we sequenced the transcriptome in L. crocea liver tissue with a Roche 454 sequencing platform and assembled the transcriptome into 93 801 transcripts. Of them, 38 856 transcripts were successfully annotated in nt, nr, Swiss-Prot, InterPro, COG, GO and KEGG databases. Based on the annotation information, 3 165 unigenes related to growth and immunity were identified. Additionally, a total of 6 391 simple sequence repeats (SSRs) were identified from the transcriptome, among which 4 498 SSRs had enough flanking regions to design primers for polymerase chain reactions (PCR). To access the polymorphism of these markers, 30 primer pairs were randomly selected for PCR amplification and validation in 30 individuals, and 12 primer pairs (40.0%) exhibited obvious length polymorphisms. This work applied RNA-Seq to assemble and analyze a live transcriptome in L. crocea. With gene annotation and sequence information, genes related to growth and immunity were identified and massive SSR markers were developed, providing valuable genetic resources for future gene functional analysis and selective breeding of L. crocea.

Molecular cloning of the heat shock protein 20 gene from Paphia textile and its expression in response to heat shock

P. textile is an important aquaculture species in China and is mainly distributed in Fujian, Guangdong, and Guangxi Provinces. In this study, an HSP20 cDNA designated PtHSP20 was cloned from P. textile. The full-length cDNA of PtHSP20 is 1 090 bp long and contains a 5′ untranslated region (UTR) of 93 bp, a 3′ UTR of 475 bp, and an open reading frame (ORF) of 522 bp. The PtHSP20 cDNA encodes 173 amino acid residues and has a molecular mass of 20.22 kDa and an isoelectric point of 6.2. Its predicted amino acid sequence shows that PtHSP20 contains a typical α-crystallin domain (residues 77–171) and three polyadenylation signal-sequences at the C-terminus. According to an amino acid sequence alignment, PtHSP20 shows moderate homology to other mollusk sHSPs. PtHSP20 mRNA was present in all of the test tissues including the heart, digestive gland, adductor muscle, gonad, gill, and mantle, with the highest concentration found in the gonad. Under the stress of high temperature, the expression of PtHSP20 mRNA was down-regulated in all of the tissues except the adductor muscle and gonad.

Thermal tolerance evaluation and related microsatellite marker screening and identification in the large yellow croaker Larimichthys crocea

Thermal tolerance to high temperature was evaluated in the large yellow croaker Larimichthys crocea. The survival thermal maximum for L. crocea was 33.0°C, the 50% critical thermal maximum (50% CTMax) was 35.5°C, and the critical thermal maximum (CTMax) was 36.0°C. Three microsatellite markers (LYC0148, LYC0200 and LYC0435), associated with thermal tolerance were screened and identified using a Bulked Segregation Analysis (BSA) method. These markers have six amplified fragments in which four are related to thermal tolerance. These fragments were cloned and sequenced, and the results showed the core motif were all “AC” repeats. For LYC0148 and LYC0200, the lengths of fragments are 181 bp and 197 bp, respectively. For LYC0435, which has two fragments, the fragment lengths are 112 bp and 100 bp. The results provide useful molecular markers for thermal-tolerance breeding of large yellow croaker in the near future.

Molecular cloning, characterization and expression analysis of a catalase gene in Paphia textile

Catalase is an important antioxidant protein that can protect organisms against various forms of oxidative damage by eliminating hydrogen peroxide. In this study, the catalase cDNA of Paphia textile (PtCAT) was cloned using RTPCR and rapid amplification of cDNA ends (RACE). PtCAT is 1 921 bp long and consists of a 5′-UTR of 50 bp, a 3′-UTR of 349 bp, and an ORF of 1 542 bp that encodes 513 amino acids with a molecular weight of 58.4 kD and an estimated isoelectric point of 8.2. Sequence alignment indicated that PtCAT contained a highly conserved catalytic signature motif (61FNRERIPERVVHAKGAG77), a proximal heme-ligand signature sequence (352RLFSYSDP359), and three catalytic amino acid residues (H72, N145, and Y356). PtCAT also contains two putative N-glycosylation sites (34NKT36 and 437NFT439) and a peroxisome-targeting signal (511AQL513). Furthermore, PtCAT shares 53%–88% identity and 29%–89% similarity with other catalase amino acid sequences. PtCAT mRNA was present in all tested organs, including the heart, digestive gland, adductor muscle, gonad, gill, and mantle, but its expression was highest in the digestive gland. High-temperature-induced stress produced two expression patterns of PtCAT mRNA: first, an initial up-regulation followed by a down-regulation in the heart, digestive gland, and gonad and, second, consistent down-regulation in all other organs. These results demonstrate that PtCAT is a typical member of the catalase family and might be involved in the responses to harmful environmental factors.