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Construction of a Normalized Full-Length cDNA Library of Cephalopod Amphioctopus fangsiao and Development of microsatellite Markers

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中国海洋大学学报（英文版） 2017年第5期 897-904页

作者： FENG Yanwei LIU Wenfen XU Xin YANG Jianmin WANG Weijun WEI Xiumei LIU Xiangquan SUN Guohua Key Laboratory of Marine Ecological Restoration Shan Dong Marine Resource and Environment Research Institute College of Fisheries and Life Science Shanghai Ocean University Key Laboratory of Marine Genetics and Breeding of Ministry of Education Ocean University of China

Amphioctopus fangsiao is one of the most economically important species and has been considered to be a candidate for aquaculture. In order to facilitate its fine-scale genetic analyses, we constructed a normalized full-length library successfully and developed a set of microsatellite markers in this study. The normalized full-length library had a storage capacity of 6.9×105 independent clones. The recombination efficiency was 95% and the average size of inserted fragments was longer than 1000 bp. A total of 3440 high quality ESTs were obtained, which were assembled into 1803 unigenes. Of these unigenes, 450(25%) were assigned into 33 Gene Ontology terms, 576(31.9%) into 153 Kyoto Encyclopedia of Genes and Genomes pathways, and 275(15.3%) into 22 Clusters of Orthologous Groups. Seventy-six polymorphic microsatellite markers were identified. The number of alleles per locus ranged from 4 to 17, and the observed and expected heterozygosities varied between 0.167 and 0.967 and between 0.326 and 0.944, respectively. Twelve loci were significantly deviated from Hardy-Weinberg equilibrium after Bonferroni correction and no linkage disequilibrium was found between different loci. This study provided not only a useful resource for the isolation of the functional genes, but also a set of informative microsatellites for the assessment of population structure and conservation genetics of A. fangsiao.

关键词： Amphioctopus fangsiao cDNA library EST microsatellite

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Molecular Grouping of Grateloupia Tissues Collected Along Chinese Coast and microsatellite Diversity Analysis of G. asiatica

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中国海洋大学学报（英文版） 2018年第4期 925-931页

作者： PENG Chong DING Haiyan TANG Zhihong GUO Li YANG Guanpin College of Marine Life Sciences，Ocean University of China Institute of Evolution & Marine Biodiversity，Ocean University of China Key Laboratory of Marine Genetics and Breeding of Chinese Ministry of Education，Ocean University of China

Genus Grateloupia is one of the most speciose genera in family Halymeniales. It is also one of the sources for natural materials, food and medicine. With different environments, Grateloupia change their morphological characteristics, making their morphological identification very difficult. In addition, few of the species diversity in this genus has been described before. In this study, phylogenetic analysis based on rbc L gene sequence was employed to group Grateloupia collected from three locations along Chinese coast. The microsatellites were also used to evaluate their genetic diversity. In total, the tissue parts of 6 putative species were collected from G. asiatica, G. livida, G. lanceolate, G. catenata, G. turuturu and G. filicina. In order to evaluate their genetic diversity and then conserve them better, 40 microsatellites available for Grateloupia were used to evaluate their genetic diversity, and 11 microsatellites were found to be applicable to determine the genetic diversity of G. asiatica. It was found that the genetic diversity of G. asiatica around Qingdao was very rich. We suggested that the species of genus Grateloupia should be identified based on rbc L phylogenetic analysis before the diversity evaluation with microsatellites. The microsatellites should be developed for each species of Grateloupia so that their genetic diversity can be evaluated appropriately.

关键词： GRATELOUPIA rbc L MOLECULAR GROUPING microsatellite genetic diversity

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microsatellites reveal strong genetic structure in the common cutworm, Spodoptera litura

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Journal of Integrative Agriculture 2019年第3期18卷 636-643页

作者： WU Huai-heng WAN Peng HUANG Min-song LEI Chao-liang Hubei Insect Resources Utilization and Sustainable Pest Management Key Laboratory Huazhong Agricultural University Key Laboratory of Integrated Pest Management on Crops in Central China Institute of Plant Protection and Soil ScienceHubei Academy of Agricultural Sciences

The common cutworm, Spodoptera litura(Lepidoptera: Noctuidae), is a voracious agricultural pest. To increase understanding of the migration patterns and genetic diversity of different geographic populations of this species, we analyzed genetic variation in nine microsatellite loci among 576 individuals collected from 17 locations in China and one in Myanmar during 2011–2012. We successfully identified 162 alleles, with an average of 18 alleles per locus and a range of 5 to 34. The mean observed heterozygosity of the 18 populations ranged from 0.18 to 0.98, and the expected heterozygosity ranged from 0.19 to 0.89. For the nine microsatellite markers studied, polymorphism information content ranged from 0.18 to 0.88(mean=0.64). We found low genetic differentiation among the 18 populations(mean F-statistics(F_(ST))=0.05) and high genetic diversity among individuals. Principle coordinates analysis indicated no significant correlation between geographic and genetic distance(r=0.04). The value of N_m(N_m>4) estimated using coalescent-based methods suggests strong gene flow with migration. The nine microsatellite markers identified in this study will be beneficial for further investigation of migration patterns and genetic diversity in S. litura.

关键词： Spodoptera litura microsatellite genetic diversity

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Identification of microsatellites in cattle unigenes

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Journal of Genetics and Genomics 2008年第5期35卷 261-266页

作者： Qiuliang Yan Yinghan Zhang Hongbin Li Caihong Wei Lili Niu Shan Guan Shangang Li Lixin Du College of Animal Science and Technology NorthwestA ＆ F University Yangling 712100 China National Center for Molecular Genetics and Breeding of Animal Institute of Animal Sciences Chinese Academy of Agricultural Sciences Beijing 100094 China Institute of Crop Sciences Chinese Academy of Agricultural Sciences Beijing 100081 China

To identify EST-SSR molecular markers, 41,986 cattle UniGene sequences from NCBI were mined for analyzing SSRs. A total of 1,831 SSRs were identified from 1,666 ESTs, which represented an average density of 19.88 kb per SSR. The frequency of EST-SSRs was 4.0%. The dinucleotide repeat motif was the most abundant SSR, accounting for 54%, followed by 22%, 13%, 7% and 4%, respectively, for tri-, hexa-, penta- and tetra-nucleotide repeats. Depending upon the length of the repeat unit, the length of microsatellites varied from 14 to 86 bp. Among the di- and tri-nucleotide repeats, AC/TG （57%） and AGC （12%） were the most abundant type. Annotation of EST-SSRs was also carried out. Three hundred primer pairs were randomly designed using Prime Premier 5.0 program and Oligo 5.0 for further experimental validation.

关键词： cattle microsatellite expressed sequence tags EST-SSR

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A Preliminary Genetic Linkage Map of Sinonovacula constricta(Lamarck, 1818) Based on microsatellites Derived from RAD Sequencing

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Journal of Ocean University of China 2018年第4期17卷 947-956页

作者： WU Xueping FENG Yanwei JIANG Hailin LIU Xiangquan PAN Ying Key Laboratory of Marine Ecological Restoration Shandong Marine Resource and Environment Research Institute College of Animal Science and Technology Guangxi University Key Laboratory of Protection and Utilization of Marine Resources Guangxi University of Nationalities

Sinonovacula constricta is one of the important economic aquaculture species in China. In this study, we constructed genetic linkage maps of S. constricta based on 300 microsatellite markers derived from RAD-seq using an F1 full-sib family. The female map contained 204 microsatellites assigned to 22 linkage groups, which covered 1529.5 cM with an average interval of 10.3 cM. The male consisted of 187 microsatellites in 19 linkage groups corresponding to the haploid chromosome number(n(28)19), which spanned 1429.3 cM with an average interval of 8.7 cM. The genome coverage was approximately 83.5% and 81.4%, respectively. An integrated map was constructed according to the common markers in parental linkage groups, which had a total length of 1683.8 cM with an average interval of 7.3 cM. The genome coverage of the integrated map was approximately 86.3%. The genetic linkage map would form the foundation for further studies on the quantitative trait loci(QTL), as well as accelerating the breeding process of this species.

关键词： linkage maps Sinonovacula constricta microsatellite marker RAD sequencing

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Phylogenetic relationships among five species of Armeniaca Scop. (Rosaceae) using microsatellites (SSRs) and capillary electrophoresis

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Journal of Forestry Research 2016年第5期27卷 1077-1083页

作者： Dali Fu Lüyi Ma Yue Qin Mengpei Liu Han Zhao Gaopu Zhu Hao Fu College of Forestry Beijing Forestry UniversityBeijing 100083 China Non-timber Forestry Research and Development Center Chinese Academy of Forestry Zhengzhou 450003 China College of Forestry Central South University of Forestry and Technology Changsha 410004 China

The genus Armeniaca Scop. is well known for its popular cultivated edible fruit trees such as Armeniaca vul- garis Lam. and ornamental flowers such as A. mume Sieb. Another species, A. cathayana Fu et al., one of six important dry fruits （kernel-using apricot）, is cultivated for its edible seeds in North China. In the present study, DNA from 70 individuals of A rmeniaca, including 38 of A. cathayana, 18 ofA. vulgaris, 12 ofA. sibirica, 1 ofA. dasycarpa and 1 of A. mume, was extracted and analyzed using microsatellites and capillary electrophoresis. For 20 polymorphic loci selected, 339 alleles and 140.7 effective alleles were detected. The number of alleles per locus ranged from 8 to 28, with an average of 16.95 alleles per locus. The observed heterozygosity （Ho） and the expected heterozygosity （He） ranged from 0.427 to 0.971 and from 0.737 to 0.912, respectively. The polymorphism information content varied from 0.708 to0.905, with an average of 0.827. Based on the genetic similarity among 70 individuals, a UPGMA was used to establish the phylogenetic relationships. The taxonomic positions among five species were clearly revealed, and A. cathayana was more closely related to A. vulgaris than to A. sibirica. The results will provide a scientific basis for research on the taxonomy, germplasm resources and breeding ofArmeniaca, especially for A. cathayana.

关键词： Armeniaca Scop. - Armeniaca cathayana microsatellite Capillary electrophoresis Phylogenetic relationship Polymorphism Simple sequence repeat

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Isolation and characterization of genic microsatellites from de novo assembly transcriptome in the bivalve Ruditapes philippinarum

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Journal of Oceanology and Limnology 2019年第3期37卷 1071-1079页

作者： SHANGGUAN Jingbo XU Anle HU Xiaowei LI Zhongbao Fujian Provincial Key Laboratory of Marine Fishery Resources and Eco-Environment Xiamen 361021China Fisheries College Jimei UniversityXiamen 361021China

The marine bivalve Ruditapes philippinarum(Veneridae)has always been an economically important aquaculture *** this study,106 831 unigenes and 2 664 SSR loci(1 locus/40 sequences)were achieved from the de novo assembly *** all the SSRs,tri-nucleotides(46.40%)was the most,followed by di-nucleotides(32.43%).Meanwhile,AAC/GTT(19.82%)was the most common SSR loci *** polymorphism detection using 32 wild *** individuals,34 polymorphic and 3 monomorphic SSR loci were screened,and the genetic index of them was *** results show that PIC of 30 polymorphic SSR loci was at medium and high levels(PIC>0.25).However,there were five SSR polymorphic loci(***871423,MG871428,MG871429,MG871434,MG871435)deviating from the Hardy-Weinberg equilibrium after the Bonferroni correction(adjusted P=0.001 471).The Na value(number of alleles per locus)ranged from 2 to *** addition,the Ho(observed heterozygosities)and He(expected heterozygosities)were 0.100 0-1.000 0 and 0.191 3-0.723 6,***,RNA-Seq was shown as a fast and cost-effective method for genic SSR development in non-model ***,the 37 loci from *** will further enrich the genetic information and advance the population conservation and restoration.

关键词： Ruditapes philippinarum transcriptome microsatellite genetic diversity

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Sequence Divergence of microsatellites and Phylogeny Analysis in Tetraploid Cotton Species and Their Putative Diploid Ancestors

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Journal of Integrative Plant Biology 2005年第12期47卷 1418-1430页

作者： Wang-Zhen GUO Dong FANG Wen-Duo YU Tian-Zhen ZHANG State Key Laboratory of Crop Genetics and Germplasm Enhancement Cotton Research Institute Nanjing Agricultural University Nanjing 210095 China

To determine the level of microsatellite sequence differences and to use the information to construct a phylogenetic relationship for cultivated tetraploid cotton （Gossypium spp.） species and their putative diploid ancestors, 10 genome-derived microsatellite primer pairs were used to amplify eight species, including two tetraploid and six diploid species, in Gossypium. A total of 92 unique amplicons were resolved using polyacrylamide gel electrophoresis. Each amplicon was cloned, sequenced, and analyzed using standard phylogenetic software. Allelic diversities were caused mostly by changes in the number of simple sequence repeat （SSR） motif repeats and only a small proportion resulted from interruption of the SSR motif within the locus for the same genome. The frequency of base substitutions was 0.5%-1.0% in different genomes, with only few indels found. Based on the combined 10 SSR flanking sequence data, the homology of A-genome diploid species averaged 98.9%, even though most of the amplicons were of the same size, and the sequence homology between G. gossypioides （Ulbr.） Standl. and three other D-genome species （G. raimondii Ulbr., G. davidsonii Kell., and G. thurberi Tod.） was 98.5%, 98.6%, and 98.5%, respectively. Phylogenetic trees of the two allotetraploid species and their putative diploid progenitors showed that homoelogous sequences from the A- and D-subgenome were still present in the polyploid subgenomes and they evolved independently. Meanwhile, homoelogous sequence interaction that duplicated loci in the polyploid subgenomes became phylogenetic sisters was also found in the evolutionary history of tetraploid cotton species. The results of the present study suggest that evaluation of SSR variation at the sequence level can be effective in exploring the evolutionary relationships among Gossypuim species.

关键词： diploid Gossypium microsatellite phylogenetic tree simple sequence repeat (SSR) tetraploid.

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Molecular identification of scallop planktonic larvae using species-specific microsatellites

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Acta Oceanologica Sinica 2008年第5期27卷 134-146页

作者： ZHAN Aibin HU Xiaoli BAO Lisui LU Wei PENG Wei WANG Mingling HU Jingjie Laboratory of Marine Genetics and Breeding College of Marine Life Sciences Ocean University of China Qingdao 266003 China

The identification of scallop larvae is essential to understand the population structure and community dynamics and to assess the potential environmental impacts caused by scallop larvae released or escaped. However, the larvae identification by morphological characteristics is notoriously difficult, mainly due to the small size （usually being less than 150 μm） and vague morphological characteristics among different scallop species. A simple and accurate molecular method was developed to identify four economically farmed scallop species, the Zhikong scallop Chlamys farreri, the noble scallop C. nobilis, the bay scallop Argopecten irradians and the Yesso scallop Mizuhopecten yessoensis. The tests used the high degree of species-specific microsatellite markers, which was specified by transferability analyses, assessed by reference individuals and evaluated by BLAST searches. The sensitivity test indicated that the species-specific microsatellites were sensitive enough for the detection of 1% -2% larvae in mixed plankton samples. Larvae collected from scallop hatcheries and their effluents and from the artificially controlled crosses were well identified to the species/hybrid level. The results demonstrated that the one-step PCR-based assay was technically simple, inexpensive and robust in identification analyses, and also less sensitive to initial quality of template DNA extracted from the ethanol-preserved samples for several years.

关键词： species identification scallop planktonic larva microsatellite hybridization

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Genetic diversity and population structure of Robinia pseudoacacia from six improved variety bases in China as revealed by simple sequence repeat markers

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Journal of Forestry Research 2022年第2期33卷 611-621页

作者： Qi Guo Sen Cao Li Dong Xiuyu Li Jiangtao Zhang Yuanshuai Zhang Zijie Zhang Yuhan Sun Cui Long Yingming Fan Chao Han Peiyao Han Xiaodi Liu Yun Li Beijing Advanced Innovation Center for Tree Breeding By Molecular Design Engineering Technology Research Center of Black Locust of National Forestry and Grassland AdministrationCollege of Biological Sciences and TechnologyNational Engineering Laboratory for Tree BreedingBeijing Forestry UniversityBeijing 100083P.R.China College of Agriculture Henan University of Science and TechnologyLuoyang 471023P.R.China Henan Academy of Forestry Zhengzhou 450008P.R.China Feixian County State Owned Daqingshan Forest Farm Linyi 273400P.R.China

Robinia pseudoacacia is an important afforesta-tion tree introduced to China in *** the present study,we examined the genetic diversity among 687 strains rep-resenting four improved varieties and two secondary prov-enances,comprising 641 clones and 46 ***-one simple sequence repeats(SSRs)were selected through segregation analysis and polymorphism characterization,and all sampled individuals were genotyped using well-characterized SSR *** excluding loci with non-neutral equilibrium,missing locus data and null alleles,we used 36 primer pairs to assess the genetic diversity of these germplasm resources,revealing vast genetic differentiation among the samples,with an average of 8.352 alleles per locus and a mean Shannon′s index of *** the popula-tion level,the partitioning of variability was assessed using analysis of molecular variance,which revealed 93%and 7%variation within and among collection sites,*** clusters were detected using structure analysis,indi-cating a degree of genetic differentiation among the six *** into the genetic diversity and structure of *** provide a theoretical basis for the con-servation,breeding and sustainable development in China.

关键词： Robinia pseudoacacia Genetic diversity Genetic differentiation microsatellite Population structure Black locust

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