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Development and Statistical Analysis of Genomic Molecular Markers of Hemitrygon bennettii
LIUXiaoru, XINGWanling, LIMingseng, ZHANGYaoren, LIPengfei, XUShengyong, XUKaida
Chin Agric Sci Bull ›› 2026, Vol. 42 ›› Issue (18) : 180-189.
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Abbreviation (ISO4): Chin Agric Sci Bull
Editor in chief: Yulong YIN
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Development and Statistical Analysis of Genomic Molecular Markers of Hemitrygon bennettii
The lack of effective molecular markers for germplasm identification and ambiguous genetic diversity characteristics severely restrict the research progress of Hemitrygon bennettii. This study aimed to establish fundamental data for germplasm identification and population genetic diversity evaluation of H. bennettii, and to provide molecular evidence for its phylogenetic analysis, genetic diversity conservation, and fishery resource management. The findings are expected to further promote the in-depth development of conservation genetics research on this species. High-throughput sequencing data and bioinformatics approaches were applied to screen genome-wide single nucleotide polymorphism (SNP) markers and analyze the population demographic history of H. bennettii. Genome-wide microsatellite loci were identified, and high-quality loci were screened for specific primer design. Furthermore, interspecific microsatellite variations between H. bennettii and H. akajei were comparatively analyzed. In addition, the complete mitochondrial genome of H. bennettii was assembled, and its genomic structure and variation characteristics were systematically analyzed. A total of 16150339 SNP loci were screened and annotated. Population demographic analysis based on genome-wide SNPs indicated that the H. bennettii population had experienced two expansion events and one bottleneck contraction event. In total, 521449 microsatellite loci were identified in the genome, with a distribution frequency of 163.02 loci per Mb. After strict screening, 116 high-quality microsatellite loci were obtained with specific primers successfully designed. Meanwhile, 19 microsatellite loci shared by H. bennettii and H. akajei with high interspecific heterogeneity were screened out. The total length of the assembled mitochondrial genome of H. bennettii was 17724 bp. A total of 49 variable sites were detected, and obvious intraspecific length heterogeneity was found in its mitochondrial genome. The SNP markers, microsatellite loci, and complete mitochondrial genome sequence obtained in this study can be effectively applied to subsequent population genetic analysis and germplasm identification of H. bennettii. This study provides reliable molecular baseline data for phylogenetic research, genetic diversity assessment, and scientific conservation and management of H. bennettii germplasm resources.
Hemitrygon bennettii / molecular marker / single nucleotide polymorphism (SNP) / microsatellite / mitochondrial genome
| [1] |
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| [2] |
|
| [3] |
|
| [4] |
|
| [5] |
|
| [6] |
|
| [7] |
|
| [8] |
李媛媛, 刘超男, 王嵘, 等. 分子标记在濒危物种保护中的应用[J]. 生物多样性, 2020, 28(3):9.
|
| [9] |
黄磊, 王义权. 微卫星分子标记在濒危动物保护遗传学研究中的应用[J]. 生物多样性, 2004, 12(5):6.
|
| [10] |
李军, 喻子牛. 单核苷酸多态性及其在海洋动物遗传研究中的应用[J]. 南方水产, 2010, 6(3):74-80.
|
| [11] |
We examined the rate and nature of mitochondrial DNA (mtDNA) mutations in humans using sequence data from 64,806 contemporary Icelanders from 2,548 matrilines. Based on 116,663 mother-child transmissions, 8,199 mutations were detected, providing robust rate estimates by nucleotide type, functional impact, position, and different alleles at the same position. We thoroughly document the true extent of hypermutability in mtDNA, mainly affecting the control region but also some coding-region variants. The results reveal the impact of negative selection on viable deleterious mutations, including rapidly mutating disease-associated 3243A>G and 1555A>G and pre-natal selection that most likely occurs during the development of oocytes. Finally, we show that the fate of new mutations is determined by a drastic germline bottleneck, amounting to an average of 3 mtDNA units effectively transmitted from mother to child.Copyright © 2024 Elsevier Inc. All rights reserved.
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| [12] |
Mitochondrial DNA (mtDNA) is a pivotal tool in molecular ecology, evolutionary and population genetics. The power of mtDNA analyses derives from a relatively high mutation rate and the apparent simplicity of mitochondrial inheritance (maternal, without recombination), which has simplified modelling population history compared to the analysis of nuclear DNA. However, in biology things are seldom simple, and advances in DNA sequencing and polymorphism detection technology have documented a growing list of exceptions to the central tenets of mitochondrial inheritance, with paternal leakage, heteroplasmy and recombination now all documented in multiple systems. The presence of paternal leakage, recombination and heteroplasmy can have substantial impact on analyses based on mtDNA, affecting phylogenetic and population genetic analyses, estimates of the coalescent and the myriad of other parameters that are dependent on such estimates. Here, we review our understanding of mtDNA inheritance, discuss how recent findings mean that established ideas may need to be re-evaluated, and we assess the implications of these new-found complications for molecular ecologists who have relied for decades on the assumption of a simpler mode of inheritance. We show how it is possible to account for recombination and heteroplasmy in evolutionary and population analyses, but that accurate estimates of the frequencies of biparental inheritance and recombination are needed. We also suggest how nonclonal inheritance of mtDNA could be exploited, to increase the ways in which mtDNA can be used in analyses.
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| [13] |
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| [14] |
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| [15] |
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| [16] |
|
| [17] |
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| [18] |
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| [19] |
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| [20] |
The enormous amount of short reads generated by the new DNA sequencing technologies call for the development of fast and accurate read alignment programs. A first generation of hash table-based methods has been developed, including MAQ, which is accurate, feature rich and fast enough to align short reads from a single individual. However, MAQ does not support gapped alignment for single-end reads, which makes it unsuitable for alignment of longer reads where indels may occur frequently. The speed of MAQ is also a concern when the alignment is scaled up to the resequencing of hundreds of individuals.We implemented Burrows-Wheeler Alignment tool (BWA), a new read alignment package that is based on backward search with Burrows-Wheeler Transform (BWT), to efficiently align short sequencing reads against a large reference sequence such as the human genome, allowing mismatches and gaps. BWA supports both base space reads, e.g. from Illumina sequencing machines, and color space reads from AB SOLiD machines. Evaluations on both simulated and real data suggest that BWA is approximately 10-20x faster than MAQ, while achieving similar accuracy. In addition, BWA outputs alignment in the new standard SAM (Sequence Alignment/Map) format. Variant calling and other downstream analyses after the alignment can be achieved with the open source SAMtools software package.http://maq.sourceforge.net.
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| [21] |
The Sequence Alignment/Map (SAM) format is a generic alignment format for storing read alignments against reference sequences, supporting short and long reads (up to 128 Mbp) produced by different sequencing platforms. It is flexible in style, compact in size, efficient in random access and is the format in which alignments from the 1000 Genomes Project are released. SAMtools implements various utilities for post-processing alignments in the SAM format, such as indexing, variant caller and alignment viewer, and thus provides universal tools for processing read alignments.http://samtools.sourceforge.net.
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| [22] |
The variant call format (VCF) is a generic format for storing DNA polymorphism data such as SNPs, insertions, deletions and structural variants, together with rich annotations. VCF is usually stored in a compressed manner and can be indexed for fast data retrieval of variants from a range of positions on the reference genome. The format was developed for the 1000 Genomes Project, and has also been adopted by other projects such as UK10K, dbSNP and the NHLBI Exome Project. VCFtools is a software suite that implements various utilities for processing VCF files, including validation, merging, comparing and also provides a general Perl API.http://vcftools.sourceforge.net
|
| [23] |
|
| [24] |
|
| [25] |
|
| [26] |
|
| [27] |
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| [28] |
The Molecular Evolutionary Genetics Analysis (MEGA) software has matured to contain a large collection of methods and tools of computational molecular evolution. Here, we describe new additions that make MEGA a more comprehensive tool for building timetrees of species, pathogens, and gene families using rapid relaxed-clock methods. Methods for estimating divergence times and confidence intervals are implemented to use probability densities for calibration constraints for node-dating and sequence sampling dates for tip-dating analyses. They are supported by new options for tagging sequences with spatiotemporal sampling information, an expanded interactive Node Calibrations Editor, and an extended Tree Explorer to display timetrees. Also added is a Bayesian method for estimating neutral evolutionary probabilities of alleles in a species using multispecies sequence alignments and a machine learning method to test for the autocorrelation of evolutionary rates in phylogenies. The computer memory requirements for the maximum likelihood analysis are reduced significantly through reprogramming, and the graphical user interface has been made more responsive and interactive for very big data sets. These enhancements will improve the user experience, quality of results, and the pace of biological discovery. Natively compiled graphical user interface and command-line versions of MEGA11 are available for Microsoft Windows, Linux, and macOS from www.megasoftware.net.
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| [29] |
Since the first investigation 25 years ago, the application of genetic tools to address ecological and evolutionary questions in elasmobranch studies has greatly expanded. Major developments in genetic theory as well as in the availability, cost effectiveness and resolution of genetic markers were instrumental for particularly rapid progress over the last 10 years. Genetic studies of elasmobranchs are of direct importance and have application to fisheries management and conservation issues such as the definition of management units and identification of species from fins. In the future, increased application of the most recent and emerging technologies will enable accelerated genetic data production and the development of new markers at reduced costs, paving the way for a paradigm shift from gene to genome-scale research, and more focus on adaptive rather than just neutral variation. Current literature is reviewed in six fields of elasmobranch molecular genetics relevant to fisheries and conservation management (species identification, phylogeography, philopatry, genetic effective population size, molecular evolutionary rate and emerging methods). Where possible, examples from the Indo-Pacific region, which has been underrepresented in previous reviews, are emphasized within a global perspective.© 2012 The Authors. Journal of Fish Biology © 2012 The Fisheries Society of the British Isles.
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| [30] |
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| [31] |
\n Two hundred and seven species of fish, mostly Australian marine fish, were sequenced (barcoded) for a 655 bp region of the mitochondrial cytochrome oxidase subunit I gene (\n cox1\n ). Most species were represented by multiple specimens, and 754 sequences were generated. The GC content of the 143 species of teleosts was higher than the 61 species of sharks and rays (47.1% versus 42.2%), largely due to a higher GC content of codon position 3 in the former (41.1% versus 29.9%). Rays had higher GC than sharks (44.7% versus 41.0%), again largely due to higher GC in the 3rd codon position in the former (36.3% versus 26.8%). Average within-species, genus, family, order and class Kimura two parameter (K2P) distances were 0.39%, 9.93%, 15.46%, 22.18% and 23.27%, respectively. All species could be differentiated by their\n cox1\n sequence, although single individuals of each of two species had haplotypes characteristic of a congener. Although DNA barcoding aims to develop species identification systems, some phylogenetic signal was apparent in the data. In the neighbour-joining tree for all 754 sequences, four major clusters were apparent: chimaerids, rays, sharks and teleosts. Species within genera invariably clustered, and generally so did genera within families. Three taxonomic groups—dogfishes of the genus\n Squalus\n, flatheads of the family Platycephalidae, and tunas of the genus\n Thunnus\n —were examined more closely. The clades revealed after bootstrapping generally corresponded well with expectations. Individuals from operational taxonomic units designated as\n Squalus\n species B through F formed individual clades, supporting morphological evidence for each of these being separate species. We conclude that\n cox1\n sequencing, or ‘barcoding’, can be used to identify fish species.\n
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| [32] |
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| [33] |
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| [34] |
A major duty of the Great Lakes Fishery Commission (GLFC), created in 1955, was the development a program of eradication or management of sea lamprey populations in the Great Lakes for the protection of the Great Lakes fishery. Beginning in the 1980s the GLFC shifted sea lamprey control to an integrated pest management model seeking to deploy control measures which target multiple life stages. Currently control efforts focus on limiting the area of infestation using barriers to migratory adults and eradication of larvae from streams using selective pesticides. Feedback on program effectiveness is obtained by trapping migratory adult lamprey at a series of index sites around the basin. The GLFC continues to support multiple research initiatives to develop additional control, improve current control measures, and further advance the sea lamprey control program. During the past six decades sea lamprey control in the Great Lakes has evolved as the research program has identified technological advances. Here we summarize the current state and recent advancements for two of the sea lamprey control program’s core elements, barriers and traps, highlight challenges to be addressed to continue the advancement of these program elements, and provide a series of research questions to spur interest within the research community. Further, because considerable information about these program elements is scattered among grey literature and technical reports, we summarize the history of barriers and traps in sea lamprey control in the included appendices to provide relevant program background to anyone interested in pursuing these research topics.
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| [35] |
The shark Squalus albicaudus, categorized by the International Union for Conservation of Nature red list as Data Deficient due to lack of minimal information for classification, is distributed throughout the Brazilian coast. High pressures such as overfishing and anthropic activities, as well as certain biological characteristics, including k strategists, comprise influential shark stocks reduction agents. However, genetic diversity, population structure, connectivity, and effective population size data are still limited for S. albicaudus, indicating the need for further studies. In this context, the genetic variability and population structure of S. albicaudus were investigated herein to test for panmixia. Samples were obtained from coasts of the Brazilian states of Pernambuco, Rio de Janeiro, and São Paulo along the species distribution range, and single nucleotide polymorphisms (SNPs) were assessed by the ddRADseq method. The findings revealed a panmitic S. albicaudus population, explained by certain life strategies, such as polyandry and migratory behavior. Based on the genomic findings reported herein, a single S. albicaudus population should be considered in the study area, indicating the need for specific management and conservation plans at the regional scale.
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| [36] |
The white-spotted bamboo shark (Chiloscyllium plagiosum) is an ecologically and commercially important marine cartilaginous fish in the Indo-Western Pacific. However, the resources of this species are declining in recent years due to habitat degradation and overfishing. Studies on genomics and genetic markers can provide valuable information for sound management and conservation of C. plagiosum. In this study, genome resequencing data of a domesticated C. plagiosum individual were generated for genomic comparison between wild and domesticated samples. Based on 281.17 Gb of clean sequencing data, the genome size of the domesticated sample was estimated as 4.99 Gb, with heterozygosity of 0.51% and repeat ratio of 74.67%, which is about 1 Gb larger than that of the wild sample (3.85 Gb with a repeat ratio of 63.53%). By using a reference-based approach, we assembled a nearly 3.45 Gb genome sequence of the domesticated sample, with the scaffold N50 of 69.45 Mb. Subsequent identification of genome-wide microsatellite markers confirmed the different abundance of repeat elements in wild and domesticated C. plagiosum. Additionally, a total of 8,703,211 single-nucleotide polymorphisms (SNPs) were detected and annotated. The demographic analysis based on identified SNPs revealed a large and constant effective population size of C. plagiosum after the last population expansion (~0.3 million years ago). The genomic data and identified genetic markers in this study can provide fundamental and useful information for further comparative genomics, evolutionary biology, and conservation genetics of C. plagiosum.
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| [37] |
刘晓茹, 李明僧, 张耀仁, 等. 大吻斜齿鲨基因组遗传资源调研分析[J]. 中国农学通报, 2025, 41(28):139-145.
大吻斜齿鲨(Scoliodon macrorhynchos)是中国近海常见的软骨鱼类，近年来其种群资源持续衰退，已被IUCN红色名录列为近危物种。为深入了解该物种并为其科学管理和保护提供依据，对其基因组遗传资源进行调研分析。利用全基因组重测序对大吻斜齿鲨基因组DNA序列进行测序，并利用重测序数据进行基因组初步组装和质量评估。在全基因组范围内识别微卫星简单序列重复位点，并计算其相对丰度。使用生物信息学软件从头组装线粒体基因组全序列，并将其与已发布的GenBank参考序列进行比较分析，识别变异位点，确定这些变异位点在基因组上的位置（蛋白质编码基因、tRNA基因、控制区），并分析非同义替换的情况。结果显示，大吻斜齿鲨基因组大小、杂合率、重复序列比例和GC含量分别为2991.63 Mb、0.46%、63.37%、44.34%。基因组初步组装长度为2482 Mb，scaffold N<sub>50</sub>为3342 bp。基于组装的基因组序列共识别出470542个微卫星位点，其相对丰度约为189.58个/Mb。从头组装获得的大吻斜齿鲨线粒体基因组全序列长度为16693 bp，与已发布的GenBank参考序列一致。线粒体全序列比较分析共检测到19个变异位点，其中16个位于蛋白质编码基因、2个位于tRNA基因、1个位于控制区。在蛋白质编码基因的变异位点中，共有4个非同义替换位点，分别位于COIII、ND4、ND5和Cyt b基因。本研究表明，大吻斜齿鲨基因组应为复杂型基因组。线粒体基因组的比较分析显示，蛋白质编码基因区域具有较高的变异性。在线粒体关键功能基因(COIII、ND4、ND5、Cyt b)中均检测到多个非同义替换位点，提示松散的净化选择可能是该物种线粒体基因组进化的主要驱动力。本研究首次构建了大吻斜齿鲨的基因组草图，开发了大量微卫星标记，揭示了其线粒体基因组变异特征，为后续开展大吻斜齿鲨的种群遗传结构解析、适应性进化研究及制定针对性生物多样性保护策略提供了重要的遗传资源和分子工具。
|
| [38] |
|
| [39] |
郑国彬, 赵虹博, 黄良敏, 等. 黄鳍棘鲷SNP标记开发与验证[J]. 热带海洋学报, 2023, 42(2):78-86.
|
| [40] |
赵蕊蕊. 条纹斑竹鲨基因组survey分析及遗传标记开发[D]. 舟山: 浙江海洋大学, 2023.
|
| [41] |
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| [42] |
Pampus belongs to the Perciformes, Stromateoidei, and Stromateidae and are widely distributed throughout the Indian Ocean and the Western Pacific. In this study, we employed next-generation sequencing to generate comprehensive genomic data for Pampus punctatissimus and Pampus echinogaster and subsequently compared the obtained genomic information with the published data of Pampus argenteus. P. echinogaster was identified to be the largest genome size (602 Mb), heterozygosity (1.38%), and repetitive sequence ratio (33.06%), while similar genome sizes between P. punctatissimus (540 Mb) and P. argenteus (550 Mb) were observed. A total of 402,921 and 628,298 SSRs were identified for P. punctatissimus and P. echinogaster, respectively. Additionally, 1,876, 638, and 394 single-copy homologous genes were identified from P. punctatissimus, P. echinogaster, and P. argenteus, respectively. The KEGG enrichment results of the unique single-copy genes to the three species were significantly different in genetic information processing, protein processing, and metabolic functions. The complete mitochondrial genomes of P. punctatissimus and P. echinogaster were successfully assembled, and the resulting phylogenetic relationships align with the currently reported taxonomic status of species in the Stromateoidei. Additionally, population size analysis using PSMC revealed that P. punctatissimus experienced one bottleneck effect, while P. echinogaster underwent two bottleneck events during the Pleistocene Glacial Epoch.
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| [43] |
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| [44] |
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| [45] |
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| [46] |
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| [47] |
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| [48] |
Microsatellite markers are widely used for genetic studies, but the relationship between microsatellite slippage mutation rate and the number of repeat units remains unclear. In this study, microsatellite distributions in the human genome are collected from public sequence databases. We observe that there is a threshold size for slippage mutations. We consider a model of microsatellite mutation consisting of point mutations and single stepwise slippage mutations. From two sets of equations based on two stochastic processes and equilibrium assumptions, we estimate microsatellite slippage mutation rates without assuming any relationship between microsatellite slippage mutation rate and the number of repeat units. We use the least squares method with constraints to estimate expansion and contraction mutation rates. The estimated slippage mutation rate increases exponentially as the number of repeat units increases. When slippage mutations happen, expansion occurs more frequently for short microsatellites and contraction occurs more frequently for long microsatellites. Our results agree with the length-dependent mutation pattern observed from experimental data, and they explain the scarcity of long microsatellites.
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| [49] |
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| [50] |
Intraspecific sequence variation in the D-loop region of mtDNA in white sturgeon (Acipenser transmontanus), a relict North American fish species, was examined in 27 individuals from populations of the Columbia and Fraser rivers. Thirty-three varied nucleotide positions were present in a 462-nucleotide D-loop sequence, amplified using the polymerase chain reaction. Bootstrapped neighbor-joining and maximum-parsimony trees of sequences from 19 haplotypes suggest that the two populations have recently diverged. This is consistent with the hypothesis that the Columbia River, a Pleistocene refugium habitat, was the source of founders for the Fraser River after the last glacial recession. On the basis of a divergence time of 10-12 thousand years ago, the estimated substitution rate of the white sturgeon D-loop region is 1.1-1.3 x 10(-7) nucleotides/site/year, which is comparable to rates for hypervariable sequences in the human D-loop region. Furthermore, the ratio of mean percent nucleotide differences in the D-loop (2.27%) to that in whole mtDNA (0.54%, as estimated from restriction-enzyme data) is 4.3, which is similar to the fourfold-to-fivefold-higher substitution rate estimated for the human D-loop. The high nucleotide substitution rate of the hypervariable region indicates that the vertebrate D-loop has potential as a genetic marker in molecular population studies.
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| [51] |
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| [52] |
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