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author:("Fu, diaoteng")
1.  An Integrated Genetic and Cytogenetic Map for Zhikong Scallop, Chlamys farreri, Based on Microsatellite Markers 
PLoS ONE  2014;9(4):e92567.
The reliability of genome analysis and proficiency of genetic manipulation requires knowledge of the correspondence between the genetic and cytogenetic maps. In the present study, we integrated cytogenetic and microsatellite-based linkage maps for Zhikong scallop, Chlamys farreri. Thirty-eight marker-anchored BAC clones standing for the 19 linkage groups were used to be FISH probes. Of 38 BAC clones, 30 were successfully located on single chromosome by FISH and used to integrate the genetic and cytogenetic map. Among the 19 linkage groups, 12 linkage groups were physically anchored by 2 markers, 6 linkage groups were anchored by 1 marker, and one linkage group was not anchored any makers by FISH. In addition, using two-color FISH, six linkage groups were distinguished by different chromosomal location; linkage groups LG6 and LG16 were placed on chromosome 10, LG8 and LG18 on chromosome 14. As a result, 18 of 19 linkage groups were localized to 17 pairs of chromosomes of C. farreri. We first integrated genetic and cytogenetic map for C. farreri. These 30 chromosome specific BAC clones in the cytogenetic map could be used to identify chromosomes of C. farreri. The integrated map will greatly facilitate molecular genetic studies that will be helpful for breeding applications in C. farreri and the upcoming genome projects of this species.
doi:10.1371/journal.pone.0092567
PMCID: PMC3976258  PMID: 24705086
2.  Genome-Wide Analysis of DNA Methylation in Five Tissues of Zhikong Scallop, Chlamys farreri 
PLoS ONE  2014;9(1):e86232.
DNA methylation plays a vital role in tissue development and differentiation in eukaryotes. Epigenetic studies have been seldom conducted in the extremely diverse and evolutionarily highly successful bilaterian lineage Mollusca. In the present study, we conducted the genome-wide profiling of DNA methylation for five tissues of a bivalve mollusc, Chlamys farreri using the methylation-sensitive amplification polymorphism (MSAP) technique. The methylation levels were quite similar among tissues, ranging from 20.9% to 21.7%. CG methylation was the dominant type (14.9%–16.5%) in the C. farreri genome, but CHG methylation also accounted for a substantial fraction of total methylation (5.1%–6.3%). Relatively high methylation diversity was observed within tissues. Methylation differentiation between tissues was evaluated and 460 tissue-specific epiloci were identified. Kidney differs from the other tissues in DNA methylation profiles. Our study presents the first look at the tissue-specific DNA methylation patterns in a bivalve mollusc and represents an initial step towards understanding of epigenetic regulatory mechanism underlying tissue development and differentiation in bivalves.
doi:10.1371/journal.pone.0086232
PMCID: PMC3891877  PMID: 24454962
3.  Large-Scale Development of Gene-Associated Single-Nucleotide Polymorphism Markers for Molluscan Population Genomic, Comparative Genomic, and Genome-Wide Association Studies 
Mollusca is the second most diverse group of animals in the world. Despite their perceived importance, omics-level studies have seldom been applied to this group of animals largely due to a paucity of genomic resources. Here, we report the first large-scale gene-associated marker development and evaluation for a bivalve mollusc, Chlamys farreri. More than 21,000 putative single-nucleotide polymorphisms (SNPs) were identified from the C. farreri transcriptome. Primers and probes were designed and synthesized for 4500 SNPs, and 1492 polymorphic markers were successfully developed using a high-resolution melting genotyping platform. These markers are particularly suitable for population genomic analysis due to high polymorphism within and across populations, a low frequency of null alleles, and conformation to neutral expectations. Unexpectedly, high cross-species transferability was observed, suggesting that the transferable SNPs may largely represent ancestral genetic variations that have been preserved differentially among subfamilies of Pectinidae. Gene annotations were available for 73% of the markers, and 65% could be anchored to the recently released Pacific oyster genome. Large-scale association analysis revealed key candidate genes responsible for scallop growth regulation, and provided markers for further genetic improvement of C. farreri in breeding programmes.
doi:10.1093/dnares/dst048
PMCID: PMC3989488  PMID: 24277739
mollusca; single-nucleotide polymorphism (SNP); transcriptome; high resolution melting (HRM); genome-wide association (GWAS)
4.  RADtyping: An Integrated Package for Accurate De Novo Codominant and Dominant RAD Genotyping in Mapping Populations 
PLoS ONE  2013;8(11):e79960.
Genetic linkage maps are indispensable tools in genetic, genomic and breeding studies. As one of genotyping-by-sequencing methods, RAD-Seq (restriction-site associated DNA sequencing) has gained particular popularity for construction of high-density linkage maps. Current RAD analytical tools are being predominantly used for typing codominant markers. However, no genotyping algorithm has been developed for dominant markers (resulting from recognition site disruption). Given their abundance in eukaryotic genomes, utilization of dominant markers would greatly diminish the extensive sequencing effort required for large-scale marker development. In this study, we established, for the first time, a novel statistical framework for de novo dominant genotyping in mapping populations. An integrated package called RADtyping was developed by incorporating both de novo codominant and dominant genotyping algorithms. We demonstrated the superb performance of RADtyping in achieving remarkably high genotyping accuracy based on simulated and real mapping datasets. The RADtyping package is freely available at http://www2.ouc.edu.cn/mollusk/ detailen.asp?id=727.
doi:10.1371/journal.pone.0079960
PMCID: PMC3836964  PMID: 24278224
5.  High-Resolution Linkage and Quantitative Trait Locus Mapping Aided by Genome Survey Sequencing: Building Up An Integrative Genomic Framework for a Bivalve Mollusc 
Genetic linkage maps are indispensable tools in genetic and genomic studies. Recent development of genotyping-by-sequencing (GBS) methods holds great promise for constructing high-resolution linkage maps in organisms lacking extensive genomic resources. In the present study, linkage mapping was conducted for a bivalve mollusc (Chlamys farreri) using a newly developed GBS method—2b-restriction site-associated DNA (2b-RAD). Genome survey sequencing was performed to generate a preliminary reference genome that was utilized to facilitate linkage and quantitative trait locus (QTL) mapping in C. farreri. A high-resolution linkage map was constructed with a marker density (3806) that has, to our knowledge, never been achieved in any other molluscs. The linkage map covered nearly the whole genome (99.5%) with a resolution of 0.41 cM. QTL mapping and association analysis congruously revealed two growth-related QTLs and one potential sex-determination region. An important candidate QTL gene named PROP1, which functions in the regulation of growth hormone production in vertebrates, was identified from the growth-related QTL region detected on the linkage group LG3. We demonstrate that this linkage map can serve as an important platform for improving genome assembly and unifying multiple genomic resources. Our study, therefore, exemplifies how to build up an integrative genomic framework in a non-model organism.
doi:10.1093/dnares/dst043
PMCID: PMC3925396  PMID: 24107803
bivalve; genome sequencing; 2b-RAD genotyping; linkage mapping; quantitative trait locus mapping
6.  Correction: Molecular Characterization of TGF-β Type I Receptor Gene (Tgfbr1) in Chlamys farreri, and the Association of Allelic Variants with Growth Traits 
PLoS ONE  2013;8(7):10.1371/annotation/75437eff-feb9-4592-baeb-ec7b98d1d212.
doi:10.1371/annotation/75437eff-feb9-4592-baeb-ec7b98d1d212
PMCID: PMC4102268
7.  Development of a Rapid and Efficient Method for Non-Lethal DNA Sampling and Genotyping in Scallops 
PLoS ONE  2013;8(7):e68096.
Non-lethal DNA sampling has long appealed to researchers studying population and conservation genetics, as it does not necessitate removing individuals permanently from their natural environment or destroying valuable samples. However, such an approach has not yet been well established in bivalves. In this study, we demonstrate that the gill represents a good source of tissue for non-lethal sampling in scallops. Removal of a few gill filaments caused no noticeable behavioral abnormalities or increased mortality rates in Zhikong scallop (Chlamys farreri) during a three-month period of observation. To facilitate rapid gill-based DNA extraction, six methods (MA-MF) were designed and evaluated, each requiring less than one hour of processing time. The optimal method was identified as MF, in terms of maintaining DNA integrity and genotyping accuracy. Further optimization of MF method by orthogonal experimental design suggested that the utilization of gills could be limited to 2 mg of sample, which is sufficient for performing up to 20,000 PCR reactions. We also demonstrate the excellent cross-species utility of MF in two additional scallop species, Yesso scallop (Patinopecten yessoensis) and bay scallop (Argopecten irradians). Taken together, our study provides a rapid and efficient approach for applying non-lethal DNA sampling in bivalve species, which would serve as a valuable tool for maintaining bivalve populations and conservation genetics, as well as in breeding studies.
doi:10.1371/journal.pone.0068096
PMCID: PMC3706602  PMID: 23874509
8.  Transcriptome Sequencing of Zhikong Scallop (Chlamys farreri) and Comparative Transcriptomic Analysis with Yesso Scallop (Patinopecten yessoensis) 
PLoS ONE  2013;8(5):e63927.
Background
Bivalves play an important role in the ecosystems they inhabit and represent an important food source all over the world. So far limited genetic research has focused on this group of animals largely due to the lack of sufficient genetic or genomic resources. Here, we performed de novo transcriptome sequencing to produce the most comprehensive expressed sequence tag resource for Zhikong scallop (Chlamys farreri), and conducted the first transcriptome comparison for scallops.
Results
In a single 454 sequencing run, 1,033,636 reads were produced and then assembled into 26,165 contigs. These contigs were then clustered into 24,437 isotigs and further grouped into 20,056 isogroups. About 47% of the isogroups showed significant matches to known proteins based on sequence similarity. Transcripts putatively involved in growth, reproduction and stress/immune-response were identified through Gene ontology (GO) and KEGG pathway analyses. Transcriptome comparison with Yesso scallop (Patinopecten yessoensis) revealed similar patterns of GO representation. Moreover, 38 putative fast-evolving genes were identified through analyzing the orthologous gene pairs between the two scallop species. More than 46,000 single nucleotide polymorphisms (SNPs) and 350 simple sequence repeats (SSRs) were also detected.
Conclusion
Our study provides the most comprehensive transcriptomic resource currently available for C. farreri. Based on this resource, we performed the first large-scale transcriptome comparison between the two scallop species, C. farreri and P. yessoensis, and identified a number of putative fast-evolving genes, which may play an important role in scallop speciation and/or local adaptation. A large set of single nucleotide polymorphisms and simple sequence repeats were identified, which are ready for downstream marker development. This transcriptomic resource should lay an important foundation for future genetic or genomic studies on C. farreri.
doi:10.1371/journal.pone.0063927
PMCID: PMC3646770  PMID: 23667690
9.  Molecular Characterization of TGF-β Type I Receptor Gene (Tgfbr1) in Chlamys farreri, and the Association of Allelic Variants with Growth Traits 
PLoS ONE  2012;7(11):e51005.
Background
Scallops are an economically important aquaculture species in Asian countries, and growth-rate improvement is one of the main focuses of scallop breeding. Investigating the genetic regulation of scallop growth could benefit scallop breeding, as such research is currently limited. The transforming growth factor beta (TGF-β) signaling through type I and type II receptors, plays critical roles in regulating cell proliferation and growth, and is thus a plausible candidate growth regulator in scallops.
Results
We cloned and characterized the TGF-β type I receptor (Tgfbr1) gene from Zhikong scallops (Chlamys farreri). The deduced amino acid sequence contains characteristic residues and exhibits the conserved structure of Tgfbr1 proteins. A high expression level of scallop Tgfbr1 was detected during early embryonic stages, whereas Tgfbr1 expression was enriched in the gonad and striated muscle in adults. A single nucleotide polymorphism (SNP, c. 1815C>T) in the 3′ UTR was identified. Scallops with genotype TT had higher growth traits values than those with genotype CC or CT in a full-sib family, and significant differences were found between genotypes CC and TT for shell length, shell height, and striated muscle weight. An expression analysis detected significantly more Tgfbr1 transcripts in the striated muscle of scallops with genotype CC compared to those with genotype TT or CT. Further evaluation in a population also revealed higher striated muscle weight in scallops with genotype TT than those with the other two genotypes. The inverse correlation between striated muscle mass and Tgfbr1 expression is consistent with TGF-β signaling having a negative effect on cell growth.
Conclusion
The scallop Tgfbr1 gene was cloned and characterized, and an SNP potentially associated with both scallop growth and Tgfbr1 expression was identified. Our results suggest the negative regulation of Tgfbr1 in scallop growth and provide a candidate marker for Zhikong scallop breeding.
doi:10.1371/journal.pone.0051005
PMCID: PMC3510168  PMID: 23209843
10.  Reference-free SNP calling: improved accuracy by preventing incorrect calls from repetitive genomic regions 
Biology Direct  2012;7:17.
Background
Single nucleotide polymorphisms (SNPs) are the most abundant type of genetic variation in eukaryotic genomes and have recently become the marker of choice in a wide variety of ecological and evolutionary studies. The advent of next-generation sequencing (NGS) technologies has made it possible to efficiently genotype a large number of SNPs in the non-model organisms with no or limited genomic resources. Most NGS-based genotyping methods require a reference genome to perform accurate SNP calling. Little effort, however, has yet been devoted to developing or improving algorithms for accurate SNP calling in the absence of a reference genome.
Results
Here we describe an improved maximum likelihood (ML) algorithm called iML, which can achieve high genotyping accuracy for SNP calling in the non-model organisms without a reference genome. The iML algorithm incorporates the mixed Poisson/normal model to detect composite read clusters and can efficiently prevent incorrect SNP calls resulting from repetitive genomic regions. Through analysis of simulation and real sequencing datasets, we demonstrate that in comparison with ML or a threshold approach, iML can remarkably improve the accuracy of de novo SNP genotyping and is especially powerful for the reference-free genotyping in diploid genomes with high repeat contents.
Conclusions
The iML algorithm can efficiently prevent incorrect SNP calls resulting from repetitive genomic regions, and thus outperforms the original ML algorithm by achieving much higher genotyping accuracy. Our algorithm is therefore very useful for accurate de novo SNP genotyping in the non-model organisms without a reference genome.
Reviewers
This article was reviewed by Dr. Richard Durbin, Dr. Liliana Florea (nominated by Dr. Steven Salzberg) and Dr. Arcady Mushegian.
doi:10.1186/1745-6150-7-17
PMCID: PMC3472322  PMID: 22682067
Next-generation sequencing; single nucleotide polymorphism; genotyping; maximum likelihood; mixed Poisson/normal model

Results 1-10 (10)