A bacterial artificial chromosome library for the Chinese alligator (Alligator sinensis)

Chinese alligator (Alligator sinensis) is a rare and endangered species endemic to China. To better understand genetic details of the Chinese alligator genomic structure, a highly redundant bacterial artificial chromosome (BAC) library was constructed. This library consists of 216,238 clones with an average insert size of about 90kb, indicating that the library contains 6.8-fold genome equivalents. Subsequently, we constructed a 516kb contig map for the Chinese alligator olfactory receptor (OR) genes, which spans nine BAC clones, and subjected the BACs to full sequencing. The sequence analysis revealed that this contig contained 16 OR functional genes and meanwhile demonstrated that the nine BACs, which constituted the contig, overlapped correctly, proving the usability of this genome library. As a result, this BAC library could provide a useful platform for physical mapping, genome sequencing or complex analysis of targeted genomic regions for this rare species.     

A Comprehensive Analysis of the Phylogeny,Genomic Organization and Expression of Immunoglobulin Light Chain Genes in Alligator sinensis,an Endangered Reptile Species

Crocodilians are evolutionarily distinct reptiles that are distantly related to lizards and are thought to be the closest relatives of birds. Compared with birds and mammals, few studies have investigated the Ig light chain of crocodilians. Here, employing an Alligator sinensis genomic bacterial artificial chromosome (BAC) library and available genome data, we characterized the genomic organization of the Alligator sinensis IgL gene loci. The Alligator sinensis has two IgL isotypes, λ and κ, the same as Anolis carolinensis. The Igλ locus contains 6 C_λ genes, each preceded by a J_λ gene, and 86 potentially functional V_λ genes upstream of (J_λ-C_λ)_n. The Igκ locus contains a single C_κ gene, 6 J_κs and 62 functional V_κs. All V_L genes are classified into a total of 31 families: 19 V_λ families and 12 V_κ families. Based on an analysis of the chromosomal location of the light chain genes among mammals, birds, lizards and frogs, the data further confirm that there are two IgL isotypes in the Alligator sinensis: Igλ and Igκ. By analyzing the cloned Igλ/κ cDNA, we identified a biased usage pattern of V families in the expressed V_λ and V_κ. An analysis of the junctions of the recombined VJ revealed the presence of N and P nucleotides in both expressed λ and κ sequences. Phylogenetic analysis of the V genes revealed V families shared by mammals, birds, reptiles and Xenopus, suggesting that these conserved V families are orthologous and have been retained during the evolution of IgL. Our data suggest that the Alligator sinensis IgL gene repertoire is highly diverse and complex and provide insight into immunoglobulin gene evolution in vertebrates.     

我国扬子鳄种群及栖息地保护现状

扬子鳄(Alligator sinensis)是我国特有的古老而珍稀的爬行动物,现阶段野生扬子鳄的分布区域进一步萎缩,成为彼此孤立的点状,残存栖息地的生态环境趋于恶化,野生鳄数量估计为120～150条,老年化程度高。扬子鳄的保护管理依据现实状况及时加以调整,加大了野外保护力度,逐步改善野生鳄的栖息生境,实施野外放归工程,初步遏制了野生鳄数量迅速下滑的局面。人工饲养种群数量已逾10 000条,当前人工饲养繁殖的重点是管理好有限的遗传多样性资源。扬子鳄的研究主要涉及形态学、解剖学、组织胚胎学、生态学、生理生化、细胞及分子生物学,人工饲养繁殖技术等方面。营养生理和保护遗传学的应用研究有待加强。     

Molecular characterization and tissue expression profiles of insulin-like growth factor binding protein-1 (IGFBP-1) in Chinese alligator <Emphasis Type="Italic">Alligator sinensis</Emphasis> during the active and hibernating periods

The Chinese alligator Alligator sinensis (Fauvel, 1879) is an endangered species endemic to China. Up to this date, very little is known about the regulation of its growth and development. Insulin-like growth factor binding protein-1 (IGFBP-1) plays essential roles in regulating vertebrate growth and development by modulating IGFs action. In this study, we cloned the full-length cDNA of Chinese alligator IGFBP-1 (caIGFBP-1) for the first time, which has an open reading frame (ORF) of 885-bp nucleotides encoding a peptide of 294 amino acid residues. The caIGFBP-1 contains all features of IGFBP-1 peptide with the conserved cysteine-rich N- and C-terminal domains and variable central L-domain. Multiple alignment analysis showed that caIGFBP-1 shares high identity in amino acid sequence with the IGFBP-1 s of American alligator Alligator mississippiensis (Daudin, 1802) (99.32%) and birds (66.32–74.53%). Phylogenetic tree analysis of IGFBP-1 amino acid sequences indicated that alligators cluster into bird branch. The caIGFBP-1 is widely expressed in all the examined tissues, the expression level is highest in liver, higher in kidney, moderate in pancreas, stomach, small intestine, and lower in ovary, muscle, lung, heart. During the hibernating period, the expression level of caIGFBP-1 was significantly reduced in liver, kidney, stomach, pancreas and small intestine, while did not significantly change in heart, lung, ovary and muscle. The caIGFBP-1 mRNA expression changes during the active and hibernating periods, which coincides with that of Chinese alligator IGF, implicating that it might play important roles in the IGF-mediated growth and development of Chinese alligator.     

Extreme Evolutionary Conservation of CaSox4 Gene* in Alligator sinensis*

The Sox genes form a gene family related by homology to the high-mobility-group (HMG) box region of the testis-determining gene, SRY.Using degenerate primer PCR, we have cloned and sequenced the HMG-box motif ofCaSox4 gene from genomic DNA of the Chinese alligator,Alligator sinensis, a species closely related to American alligator, Alligator mississippiensis. Sequence comparison showed that CaSox4 gene shared 97% homology at the nucleotide level and complete identity at the amino acid level, respectively, withAES4 gene of A. mississippiensis. It indicates that the Sox4gene has been extremely conserved during evolution. The result obtained in the present study strongly suggests that theSox4 gene did not diverge during the differentiation of two closely related alligators, which are thought to have originated from a common ancestor and have existed largely unchanged for 200–250 Myr.     

Genomic investigation of the Chinese alligator reveals wild-extinct genetic diversity and genomic consequences of their continuous decline

Critically endangered species are usually restricted to small and isolated populations. High inbreeding without gene flow among populations further aggravates their threatened condition and reduces the likelihood of their long-term survival. Chinese alligator (Alligator sinensis) is one of the most endangered crocodiles in the world and has experienced a continuous decline over the past c. 1 million years. In order to identify the genetic status of the remaining populations and aid conservation efforts, we assembled the first high-quality chromosome-level genome of Chinese alligator and explored the genomic characteristics of three extant breeding populations. Our analyses revealed the existence of at least three genetically distinct populations, comprising two breeding populations in China (Changxing and Xuancheng) and one breeding population in an American wildlife refuge. The American population does not belong to the last two populations of its native range (Xuancheng and Changxing), thus representing genetic diversity extinct in the wild and provides future opportunities for genetic rescue. Moreover, the effective population size of these three populations has been continuously declining over the past 20 ka. Consistent with this decline, the species shows extremely low genetic diversity, a large proportion of long runs of homozygous fragments, and mutational load across the genome. Finally, to provide genomic insights for future breeding management and conservation, we assessed the feasibility of mixing extant populations based on the likelihood of introducing new deleterious alleles and signatures of local adaptation. Overall, this study provides a valuable genomic resource and important genomic insights into the ecology, evolution, and conservation of critically endangered alligators.     

Genome evolution in Reptilia: <Emphasis Type="Italic">in silico</Emphasis> chicken mapping of 12,000 BAC-end sequences from two reptiles and a basal bird

Background

With the publication of the draft chicken genome and the recent production of several BAC clone libraries from non-avian reptiles and birds, it is now possible to undertake more detailed comparative genomic studies in Reptilia. Of interest in particular are the genomic events that transformed the large, repeat-rich genomes of mammals and non-avian reptiles into the minimalist chicken genome. We have used paired BAC end sequences (BESs) from the American alligator (Alligator mississippiensis), painted turtle (Chrysemys picta) and emu (Dromaius novaehollandiae) to investigate patterns of sequence divergence, gene and retroelement content, and microsynteny between these species and chicken.

Results

From a total of 11,967 curated BESs, we successfully mapped 725, 773 and 2597 sequences in alligator, turtle, and emu, respectively, to sites in the draft chicken genome using a stringent BLAST protocol. Most commonly, sequences mapped to a single site in the chicken genome. Of 1675, 1828 and 2936 paired BESs obtained for alligator, turtle, and emu, respectively, a total of 34 (alligator, 2%), 24 (turtle, 1.3%) and 479 (emu, 16.3%) pairs were found to map with high confidence and in the correct orientation and with BAC-sized intermarker distances to single chicken chromosomes, including 25 such paired hits in emu mapping to the chicken Z chromosome. By determining the insert sizes of a subset of BAC clones from these three species, we also found a significant correlation between the intermarker distance in alligator and turtle and in chicken, with slopes as expected on the basis of the ratio of the genome sizes.

Conclusion

Our results suggest that a large number of small-scale chromosomal rearrangements and deletions in the lineage leading to chicken have drastically reduced the number of detected syntenies observed between the chicken and alligator, turtle, and emu genomes and imply that small deletions occurring widely throughout the genomes of reptilian and avian ancestors led to the ~50% reduction in genome size observed in birds compared to reptiles. We have also mapped and identified likely gene regions in hundreds of new BAC clones from these species.

     

The karyotype of Alligator mississippiensis,and chromosomal mapping of the ZFY/X homologue,Zfc

Comparative mapping studies of X-linked genes in mammals have provided insights into the evolution of the X chromosome. Many reptiles including the American alligator, Alligator mississippiensis, do not appear to possess heteromorphic sex chromosomes, and sex is determined by the incubation temperature of the egg during embryonic development. Mapping of homologues of mammalian X-linked genes in reptiles could lead to a greater understanding of the evolution of vertebrate sex chromosomes. One of the genes used in the mammalian mapping studies was ZFX, an X-linked copy of the human ZFY gene which was originally isolated as a candidate for the mammalian testis-determining factor (TDF). ZFX is X-linked in eutherians, but maps to two autosomal locations in marsupials and monotremes, close to other genes associated with the eutherian X. The alligator homologue of the ZFY/ZFX genes, Zfc, has been isolated and described previously. A detailed karyotype of A. mississippiensis is presented, together with chromosomal in situ hybridisation data localising the Zfc gene to chromosome 3. Further chromosomal mapping studies using eutherian X-linked genes may reveal conserved chromosomal regions in the alligator that have become part of the eutherian X chromosome during evolution.     

The first complete mitochondrial genome from Bostrychus genus (Bostrychus sinensis) and partitioned Bayesian analysis of Eleotridae fish phylogeny

To understand the phylogenetic position of Bostrychus sinensis in Eleotridae and the phylogenetic relationships of the family, we determined the nucleotide sequence of the mitochondrial (mt) genome of Bostrychus sinensis. It is the first complete mitochondrial genome sequence of Bostrychus genus. The entire mtDNA sequence was 16508 bp in length with a standard set of 13 protein-coding genes, 22 transfer RNA genes (tRNAs), two ribosomal RNA genes (rRNAs) and a noncoding control region. The mitochondrial genome of B. sinensis had common features with those of other bony fishes with respect to gene arrangement, base composition, and tRNA structures. Phylogenetic hypotheses within Eleotridae fish have been controversial at the genus level. We used the mitochondrial cytochrome b (cytb) gene sequence to examine phylogenetic relationships of Eleotridae by using partitioned Bayesian method. When the specific models and parameter estimates were presumed for partitioning the total data, the harmonic mean –lnL was improved. The phylogenetic analysis supported the monophyly of Hypseleotris and Gobiomorphs. In addition, the Bostrychus were most closely related to Ophiocara, and the Philypnodon is also the sister to Microphlypnus, based on the current datasets. Further, extensive taxonomic sampling and more molecular information are needed to confirm the phylogenetic relationships in Eleotridae.     

Eleven novel microsatellite markers for the Chinese alligator (Alligator sinensis)

Chinese alligator (Alligator sinensis) is a critically endangered species endemic to China. In this study, we developed 11 novel microsatellite loci for this rare species and applied them to examine genetic variation of indigenous alligators from Changxing Nature Reserve and America-born Chinese alligators. The 11 polymorphic microsatellites presented a total of 31 alleles among 57 individuals scored, yielding an average of 2.82 alleles per locus. One allele was unique to the American population but four private alleles were detected in the Changxing population. The average expected and observed heterozygosities were 0.400 and 0.482 for the Changxing alligators and 0.520 and 0.621 for the America-born individuals, respectively. These microsatellite markers would be useful tools in the genetic examination of this endangered species.     

Multi-Platform Next-Generation Sequencing of the Domestic Turkey (Meleagris gallopavo): Genome Assembly and Analysis

A synergistic combination of two next-generation sequencing platforms with a detailed comparative BAC physical contig map provided a cost-effective assembly of the genome sequence of the domestic turkey (Meleagris gallopavo). Heterozygosity of the sequenced source genome allowed discovery of more than 600,000 high quality single nucleotide variants. Despite this heterozygosity, the current genome assembly (∼1.1 Gb) includes 917 Mb of sequence assigned to specific turkey chromosomes. Annotation identified nearly 16,000 genes, with 15,093 recognized as protein coding and 611 as non-coding RNA genes. Comparative analysis of the turkey, chicken, and zebra finch genomes, and comparing avian to mammalian species, supports the characteristic stability of avian genomes and identifies genes unique to the avian lineage. Clear differences are seen in number and variety of genes of the avian immune system where expansions and novel genes are less frequent than examples of gene loss. The turkey genome sequence provides resources to further understand the evolution of vertebrate genomes and genetic variation underlying economically important quantitative traits in poultry. This integrated approach may be a model for providing both gene and chromosome level assemblies of other species with agricultural, ecological, and evolutionary interest.     

Use of alligator hole abundance and occupancy rate as indicators for restoration of a human-altered wetland

Use of indicator species as a measure of ecosystem conditions is an established science application in environmental management. Because of its role in shaping wetland systems, the American alligator (Alligator mississippiensis) is one of the ecological indicators for wetland restoration in south Florida, USA. We conducted landscape-level aerial surveys of alligator holes in two different habitats in a wetland where anthropogenic modification of surface hydrology has altered the natural system. Alligator holes were scarcer in an area where modified hydrology caused draining and frequent dry-downs compared to another area that maintains a functional wetland system. Lower abundance of alligator holes indicates lack of alligator activities, lower overall species diversity, and lack of dry-season aquatic refugia for other organisms. The occupancy rate of alligator holes was lower than the current restoration target for the Everglades, and was variable by size class with large size-class alligators predominantly occupying alligator holes. This may indicate unequal size-class distribution, different habitat selection by size classes, or possibly a lack of recruitment. Our study provides pre-restoration baseline information about one indicator species for the Everglades. Success of the restoration can be assessed via effective synthesis of information derived by collective research efforts on the entire suite of selected ecological indicators.     

Genome-Wide Comparative Analysis Reveals Similar Types of NBS Genes in Hybrid Citrus sinensis Genome and Original Citrus clementine Genome and Provides New Insights into Non-TIR NBS Genes

In this study, we identified and compared nucleotide-binding site (NBS) domain-containing genes from three Citrus genomes (C. clementina, C. sinensis from USA and C. sinensis from China). Phylogenetic analysis of all Citrus NBS genes across these three genomes revealed that there are three approximately evenly numbered groups: one group contains the Toll-Interleukin receptor (TIR) domain and two different Non-TIR groups in which most of proteins contain the Coiled Coil (CC) domain. Motif analysis confirmed that the two groups of CC-containing NBS genes are from different evolutionary origins. We partitioned NBS genes into clades using NBS domain sequence distances and found most clades include NBS genes from all three Citrus genomes. This suggests that three Citrus genomes have similar numbers and types of NBS genes. We also mapped the re-sequenced reads of three pomelo and three mandarin genomes onto the C. sinensis genome. We found that most NBS genes of the hybrid C. sinensis genome have corresponding homologous genes in both pomelo and mandarin genomes. The homologous NBS genes in pomelo and mandarin suggest that the parental species of C. sinensis may contain similar types of NBS genes. This explains why the hybrid C. sinensis and original C. clementina have similar types of NBS genes in this study. Furthermore, we found that sequence variation amongst Citrus NBS genes were shaped by multiple independent and shared accelerated mutation accumulation events among different groups of NBS genes and in different Citrus genomes. Our comparative analyses yield valuable insight into the structure, organization and evolution of NBS genes in Citrus genomes. Furthermore, our comprehensive analysis showed that the non-TIR NBS genes can be divided into two groups that come from different evolutionary origins. This provides new insights into non-TIR genes, which have not received much attention.     

Megabase Level Sequencing Reveals Contrasted Organization and Evolution Patterns of the Wheat Gene and Transposable Element Spaces

To improve our understanding of the organization and evolution of the wheat (Triticum aestivum) genome, we sequenced and annotated 13-Mb contigs (18.2 Mb) originating from different regions of its largest chromosome, 3B (1 Gb), and produced a 2x chromosome survey by shotgun Illumina/Solexa sequencing. All regions carried genes irrespective of their chromosomal location. However, gene distribution was not random, with 75% of them clustered into small islands containing three genes on average. A twofold increase of gene density was observed toward the telomeres likely due to high tandem and interchromosomal duplication events. A total of 3222 transposable elements were identified, including 800 new families. Most of them are complete but showed a highly nested structure spread over distances as large as 200 kb. A succession of amplification waves involving different transposable element families led to contrasted sequence compositions between the proximal and distal regions. Finally, with an estimate of 50,000 genes per diploid genome, our data suggest that wheat may have a higher gene number than other cereals. Indeed, comparisons with rice (Oryza sativa) and Brachypodium revealed that a high number of additional noncollinear genes are interspersed within a highly conserved ancestral grass gene backbone, supporting the idea of an accelerated evolution in the Triticeae lineages.     

扬子鳄分子遗传学与遗传多样性研究现状

扬子鳄 (Alligatorsinensis)是中国特有的珍稀物种 ,其遗传资源的保护已受到广泛关注和重视。本文简要介绍了近年来有关扬子鳄在分子系统学、遗传多样性与种群遗传结构、线粒体基因组、个体识别以及性别决定方面的分子遗传学研究状况。