Xiaoting Huang

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.

Scallop genome provides insights into evolution of bilaterian karyotype and development

Reconstructing the genomes of bilaterian ancestors is central to our understanding of animal evolution, where knowledge from ancient and/or slow-evolving bilaterian lineages is critical. Here we report a high-quality, chromosome-anchored reference genome for the scallop Patinopecten yessoensis, a bivalve mollusc that has a slow-evolving genome with many ancestral features. Chromosome-based macrosynteny analysis reveals a striking correspondence between the 19 scallop chromosomes and the 17 presumed ancestral bilaterian linkage groups at a level of conservation previously unseen, suggesting that the scallop may have a karyotype close to that of the bilaterian ancestor. Scallop Hox gene expression follows a new mode of subcluster temporal co-linearity that is possibly ancestral and may provide great potential in supporting diverse bilaterian body plans. Transcriptome analysis of scallop mantle eyes finds unexpected diversity in phototransduction cascades and a potentially ancient Pax2/5/8-dependent pathway for noncephalic eyes. The outstanding preservation of ancestral karyotype and developmental control makes the scallop genome a valuable resource for understanding early bilaterian evolution and biology.

RADtyping: An Integrated Package for Accurate De Novo Codominant and Dominant RAD Genotyping in Mapping Populations

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.

Scallop genome reveals molecular adaptations to semi-sessile life and neurotoxins

Bivalve molluscs are descendants of an early-Cambrian lineage superbly adapted to benthic filter feeding. Adaptations in form and behavior are well recognized, but the underlying molecular mechanisms are largely unknown. Here, we investigate the genome, various transcriptomes, and proteomes of the scallop Chlamys farreri, a semi-sessile bivalve with well-developed adductor muscle, sophisticated eyes, and remarkable neurotoxin resistance. The scallop’s large striated muscle is energy-dynamic but not fully differentiated from smooth muscle. Its eyes are supported by highly diverse, intronless opsins expanded by retroposition for broadened spectral sensitivity. Rapid byssal secretion is enabled by a specialized foot and multiple proteins including expanded tyrosinases. The scallop uses hepatopancreas to accumulate neurotoxins and kidney to transform to high-toxicity forms through expanded sulfotransferases, probably as deterrence against predation, while it achieves neurotoxin resistance through point mutations in sodium channels. These findings suggest that expansion and mutation of those genes may have profound effects on scallop’s phenotype and adaptation.Bivalve molluscs have evolved various characteristics to adapt to benthic filter-feeding. Here, Li et al investigate the genome, transcriptomes and proteomes of scallop Chlamys farreri, revealing evidences of molecular adaptations to semi-sessile life and neurotoxins.

Long Non-Coding RNAs (lncRNAs) of Sea Cucumber: Large-Scale Prediction, Expression Profiling, Non-Coding Network Construction, and lncRNA-microRNA-Gene Interaction Analysis of lncRNAs in Apostichopus japonicus and Holothuria glaberrima During LPS Challenge and Radial Organ Complex Regeneration

Long non-coding RNA (lncRNA) structurally resembles mRNA but cannot be translated into protein. Although the systematic identification and characterization of lncRNAs have been increasingly reported in model species, information concerning non-model species is still lacking. Here, we report the first systematic identification and characterization of lncRNAs in two sea cucumber species: (1) Apostichopus japonicus during lipopolysaccharide (LPS) challenge and in heathy tissues and (2) Holothuria glaberrima during radial organ complex regeneration, using RNA-seq datasets and bioinformatics analysis. We identified A. japonicus and H. glaberrima lncRNAs that were differentially expressed during LPS challenge and radial organ complex regeneration, respectively. Notably, the predicted lncRNA-microRNA-gene trinities revealed that, in addition to targeting protein-coding transcripts, miRNAs might also target lncRNAs, thereby participating in a potential novel layer of regulatory interactions among non-coding RNA classes in echinoderms. Furthermore, the constructed coding-non-coding network implied the potential involvement of lncRNA-gene interactions during the regulation of several important genes (e.g., Toll-like receptor 1 [TLR1] and transglutaminase-1 [TGM1]) in response to LPS challenge and radial organ complex regeneration in sea cucumbers. Overall, this pioneer systematic identification, annotation, and characterization of lncRNAs in echinoderm pave the way for similar studies and future genetic, genomic, and evolutionary research in non-model species.

Comparative Cytogenetics Analysis of Chlamys farreri, Patinopecten yessoensis, and Argopecten irradians with C0t-1 DNA by Fluorescence In Situ Hybridization

The chromosomes of Chlamys farreri, Patinopecten yessoensis, and Argopecten irradians were studied by FISH using C. farreri C0t-1 DNA probes. The results showed that C0t-1 DNA signals spread on all chromosomes in the three scallops, whereas signal density and intensity were different strikingly. Clustering brighter signals presented in the centromeric and telomeric regions of most C. farreri chromosomes, and in the centromeric or pericentromeric regions of several P. yessoensis chromosomes. Comparative analysis of the mapping indicated a relatively higher homology in the repetitive DNA sequences of the genome between C. farreri and P. yessoensis than that between C. farreri and A. irradians. In addition, FISH showed that the distribution of C0t-1 DNA clustering signals in C. farreri displayed completely similar signal bands between homologous chromosomes. Based on the C0t-1 DNA fluorescent bands, a more exact karyotype of C. farreri has been obtained. In this study, the comparative analysis based on C0t-1 DNA provides a new insight into the chromosomal reconstructions during the evolution process, and it is helpful for understanding an important source of genomic diversity, species relationships, and genome evolution.

Mapping toll-like receptor signaling pathway genes of Zhikong scallop (Chlamys farreri) with FISH

Toll-like receptor (TLR) signaling pathway plays a pivotal role in the innate immune system. Studies on TLR signaling pathway genes in Zhikong scallop (Chlamys farreri) have mainly focused on sequence analysis and expression profiling, no research has been carried out on their localization. The chromosomal position of TLR signaling pathway genes can be valuable for assemblying scallop genome and analysizing gene regulatory networks. In the present study, five key TLR signaling pathway genes (CfTLR, CfMyd88, CfTRAF6, CfNFκB, and CfIκB) containing bacterial artificial chromosomes (BACs) were isolated and physically mapped through fluorescence in situ hybridization on five non-homologous chromosome pairs, showing a similar distribution to another five model species. The isolation and mapping of these key immune genes of C. farreri will aid to the research on innate immunity, assignment of interested genes to chromosomes, and integration of physical, linkage and cytogenetic maps of this species.

Genomic characterization of interspecific hybrids between the scallops Argopecten purpuratus and A. irradians irradians.

The Peruvian scallop (Argopecten purpuratus) has been introduced to China and has successfully been hybridized with the bay scallop (A. irradians irradians). The F1 hybrids of these two scallops exhibited a large increase in production traits and some other interesting new characteristics. To understand the genetic basis of this heterosis, nuclear gene and partial mtDNA sequences, and genomic in situ hybridization (GISH) were employed to analyze the genomic organization of the hybrids. Amplification of the ribosomal DNA internal transcribed spacer (ITS) showed that the parental ITS sequences were present in all the hybrid individuals, illustrating that the hybrid offspring inherited nuclear DNA from both parents. Sequence analyses of the ITS region further confirmed that the hybrids harbored alleles from their parents; some recombinant variants were also detected, which revealed some alterations in the nuclear genetic material of the hybrids. The analysis of mitochondrial 16S rDNA showed that the hybrids possessed sequences that were identical to the 16S rDNA of the female parents, proving a matrilineal inheritance of mitochondrial genes in scallops. In addition, GISH clearly discriminated between the parental chromosomes and indicated a combination of haploid genomes of duplex parents in the hybrids. The genetic analyses in our study illustrated that the F1 hybrids inherited nuclear material from both parents and cytoplasmic genetic material maternally, and some variations occurred in the genome, which might contribute to a further understanding of crossbreeding and heterosis in scallop species.

GROWTH AND REPRODUCTIVE PERFORMANCE OF TRIPLOID YESSO SCALLOPS (PATINOPECTEN YESSOENSIS) INDUCED BY HYPOTONIC SHOCK

ABSTRACT The successful induction of triploid embryos or larvae has been performed in Patinopecten yessoensis during the past 2 decades. However, no research has been reported about the performance of triploid P. yessoensis cultured in the field. This study induced triploidy in P. yessoensis by hypotonic shock and compared the growth and reproductive performance of triploids and diploids reared under commercial conditions for up to 24 mo. The main results of this study are as follows: Triploid scallops were smaller in size and weight compared with diploids and had a retarded absolute growth rate (AGR). After 24 mo of cultivation, the mean shell height, shell length, shell width, and body weight of triploids were 9%, 10%, 9%, and 25% less than diploids, respectively (P < 0.01). Although normal in sex ratio, the reproductive potential of triploids was significantly reduced. Only 87% of the triploids exhibited sex-discernible gonads during the breeding season. None of the male triploids spawned, and the percentage of female spawners among the triploid population was only 27% of that for the diploid population. The relative fecundity of triploid females was only 4% of diploid females. Triploid eggs produced mostly aneuploid larvae and had an extremely small chance of generating viable offspring when fertilized by sperm from diploid males. Most aneuploid larvae died before the D-shaped stage, and no survival exceeded 7 days. The potential to yield viable offspring from the triploid population was estimated to be only 4% × 10-6 of that of the diploid population. Despite the growth disadvantage of triploids, this study may support, in part, the energy reallocation hypothesis, because triploid AGR was similar to diploid AGR (2% variance) during the sexual maturation season. Our results also indicate that there would be no growth advantage, but instead a disadvantage, for triploid P. yessoensis growing at the experiment site. In addition, this research provides the first evidence that viable triploid molluscs can be induced by hypotonic shock, of which the practical and evolutionary implications are also discussed.

A Genome-Wide Association Study Identifies the Genomic Region Associated with Shell Color in Yesso Scallop, Patinopecten yessoensis

The shell color polymorphism widely exists in economic shellfish, which not only results in a better visual perception but also shows great value as an economic trait for breeding. Small numbers of reddish-orange shell Yesso scallops, Patinopecten yessoensis, were found in cultured populations compared to the brown majority. In this study, a genome-wide association study was conducted to understand the genetic basis of shell color. Sixty-six 2b-RAD libraries with equal numbers of reddish-orange and brown shell individuals were constructed and sequenced using the Illumina HiSeq 2000 platform. A total of 322,332,684 high-quality reads were obtained, and the average sequencing depth was 18.4×. One genomic region on chromosome 11 that included 239 single-nucleotide polymorphisms (SNPs) was identified as significantly associated with shell color. After verification by high-resolution melting in another population, two SNPs were selected as specific loci for reddish-orange shell color. These two SNPs could be used to improve the selective breeding progress of true-breeding strains with complete reddish-orange scallops. In addition, within the significantly associated genomic region, candidate genes were identified using marker sequences to search the draft genome of Yesso scallop. Three genes (LDLR, FRIS, and FRIY) with known functions in carotenoid metabolism were identified. Further study using high-performance liquid chromatography proved that the relative level of carotenoids in the reddish-orange shells was 40 times higher than that in the brown shells. These results suggested that the accumulation of carotenoids contributes to the formation of reddish-orange shells.