Genetic Polymorphism in GDF8 Region of HU Sheep Based on Microsatellite DNA

[ Objective ] This study was to analyze the genetic polymorphism in GDF8 Region of HU sheep. [Method] Four microsatellite loci including BMS1591, TEXAN-2, FCB128 and BM81124 mapped on GDF8 region of chromosome No. 2 of sheep that may be correlated with growth performance were chosen to detect the molecular genetics foundation of growth performance of Hu sheep. [ Result] Four microsatellite loci detected were high in heterozygosity, more in effective alleles number and rich in polymorphic information, all the three indices passed through the high polymorphic level （PIC 〉0.5）. [ Conclusion ] The four microsatellite loci detected could be used to estimate the genetic polymorphism of growth performance of Hu sheep.

Cloning, Characterization, and FISH Mapping of Four Satellite DNAs from Black Muntjac (Muntiacus crinifrons) and Fea’s Muntjac (M. feae)

Recent molecular cytogenetic studies demonstrate that extensive centromere-telomere fusions are the main chromosomal rearrangements underlying the karyotypic evolution of extant muntjacs. Although the molecular mechanism of tandem fusions remains unknown, satellite DNA is believed to have facilitated chromosome fusions by non-allelic homologous recombination. Previous studies detected non-random hybridization signals of cloned satellite DNA at the postulated fusion sites on the chromosomes in Indian and Chinese muntjacs. But the genomic distribution and organization of satellite DNAs in other muntjacs have not been investigated. In this study, we have isolated four satellite DNA clones （BMCS, BM700, BM 1.1 k and FM700） from the black muntjac （Muntiacus crinifrons） and Fea＇s muntjac （M. feae）, and hybridized these four clones onto chromosomes of four muntjac species （M. reevesi, M. crinifrons, M. gongshanenisis and M. feae）. Besides the predominant centromeric signals, non-random interstitial hybridization signals from satellite I and II DNA clones （BMC5, BM700 and FM700） were also observed on the arms of chromosomes of these four muntjacs. Our results provide additional support for the notion that the karyotypes of M. crinifrons, M. feae and M. gongshanensis have evolved from a 2n = 70 ancestral karyotype by a series of chromosome fusions.

Microsatellite instability and expression of DNA mismatch repair genes in malignant astrocytic tumors from adult and pediatric patients

Microsatellite instability (MSI) is used as a molecular marker for defective DNA mismatch repair (MMR) genes.We report here alterations of MSI in 15 malignant astrocytomas (WHO grade Ⅲ) and glioblastomas (GBM; WHO grade Ⅳ) of pediatric patients (2-21 years) and 12 GBM from adults (44-68 years) by comparative analysis of BAT25/BAT26 loci and 10 other microsatellite markers. High-level microsatellite instability (MSI-H) occurred in 4 of the 15 pediatric cases (26.7%) and in 1 of the 12 adult GBM cases (8.3%). Low-level mi-

Molecular phylogenetics and population demographic history of Amphioctopus fangsiao,inferred from mitochondrial and microsatellite DNA markers

Amphioctopus fangsiao(Cephalopoda:Octopodidae)is an important commercial species in the coastal waters of China.In recent years,however,the resource of A.fangsiao have declined because of habitat destruction and overfishing.To analyze the genetic variations of A.fangsiao caused by the fluctuation of resources,the population genetic structure of nine sampling locations collected from the Bohai Sea to the South China Sea were investigated,using mtDNA COI fragments and microsatellite DNA.The results of F-statistics,AMOVA,STRUCTURE and PCA analyses showed three phylogeographic clades(Clades A,B and C),revealing limited genetic exchange between north and south populations.These clades diverged in 2.23(Clades A and B)and 3.67(Clades A,B and C)million years ago,during the dramatic environmental fluctuations,such as sea level and temperature changes,have exerted great influence on the survival distribution pattern of global organisms.Our results for low genetic connectivity among A.fangsiao populations provide insights into the development of management strategies,that is,to manage this species as separate management unit.

Construction Fractional Genomic Libraries and Screening Microsatellites DNA of Esox reieherti Dybowski

Esox reieherti Dybowsk genomic microsateUites were developed by using enrichment protocols combined with radioactive hybridization protocol. Four hundred to nine hundred base pair fragments were selected for the whole genome. DNA PCR amplification after digestion with restriction endonuclease Sau 3A Ⅰ, and （CA）12, （GA）12 probes marked with biotin were used for microsateUite DNA enrichment. The product fragments were connected with carder pGEM-T and transferred into DH5α Escherichia coli competent cells, and radioactive isotope probes marked with γ^-32 p were used for the second hybridization. As a result, a total of 1600 bacteria were obtained in the microsatellite genomic libraries, positive clones accounted for 90.91% before hybridization and 81.25% after hybridization, amounting to 1300. One hundred and ninety-six positive clones were selected for sequencing, and 192 clones included microsateUite sequences. The microsateUite sequences obtained, mono-nucleotide, quad-nucleotide and quint-nucleotide repeat motifs were observed beside double-base-pairs CA/GT, GA/CT. Seventy primers were designed according to the flanking sequences by using software Primer Premier 5.0, and 32 primers were selected to be synthesized. After optimizing PCR reaction conditions, 28 primers were amplified and produced clear purpose bands. The aim of our research was to promote the development and utilization of E. reieherti genomic resource, and lay the foundation for optimizing E. reieherti breeding strain in order to detect the genetic diversity and construct a genetic map.

Sequences Characterization of Microsatellite DNA Sequences in Pacific Abalone(Haliotis discus hannai)

The microsatellite-enriched library was constructed using magnetic bead hybridization selection method, and the mi-crosatellite DNA sequences were analyzed in Pacific abalone Haliotis discus hannai. Three hundred and fifty white colonies were screened using PCR-based technique, and 84 clones were identified to potentially contain microsatellite repeat motif. The 84 clones were sequenced, and 42 microsatellites and 4 minisatellites with a minimum of five repeats were found (13.1% of white colonies screened). Besides the motif of CA contained in the oligoprobe, we also found other 16 types of microsatellite repeats including a dinucleotide repeat, two tetranucleotide repeats, twelve pentanucleotide repeats and a hexanucleotide repeat. According to Weber (1990), the microsatellite sequences obtained could be categorized structurally into perfect repeats (73.3%), imperfect repeats (13.3%), and compound repeats (13.4%). Among the microsatellite repeats, relatively short arrays (< 20 repeats) were most abundant, accounting for 75.0%. The largest length of microsatellites was 48 repeats, and the average number of repeats was 13.4. The data on the composition and length distribution of microsatellites obtained in the present study can be useful for choosing the repeat motifs for microsatellite isolation in other abalone species.

Genetic Structure and Diversity of Parental Cultivars Involved in China Mainland Sugarcane Breeding Programs as Inferred from DNA Microsatellites

To understand genetic structure and diversity of parental cultivars involved in China Mainland sugarcane breeding programs, 92 elite parents and 4 wild relatives were genotyped with 18 microsatellite DNA markers. The genetic similarity （GS） values among the cultivars ranged from 0.346 to 0.960 with an average of 0.533. Among the introduced cultivars, India accessions had the closest genetic distance to China Mainland accessions （0.447）, while Australia accessions have the furthest distance （0.503）. A comparison of allelic diversity among geographical origins showed that there were 22 China Mainland specific alleles, of which 28% were derived from native S. spontaneaum germplasm in China. Model-based genetic structure, clustering, and principal components analyses consistently revealed there were five groups within the 96 accessions. Groups 1, 2, 4, and 5 consisted of all cultivars and group 3 only contained wild germplasm. Group 2 was characterized as the Introduction group with 46 cultivars predominantly introduced from Australia, Taiwan of China, India, and USA. Groups 1, 4, and 5 consisted of cultivars mostly originated from China Mainland, defined as the Complex group, Yacheng lines group, and F134/CP72-1210 group, respectively, upon their pedigree. By understanding the genetic relationships among the parental cultivars, breeders can gain a rational basis for expanding the gene pool and select the best parental accessions for crossing.

Forecast of the Heterosis of Imported Meat Sheep by Genetic Polymorphism of Microsatellite DNA

Forecast of the heterosis of Small Tail Han sheep crossed with imported meat sheep by genetic polymorphism of microsatellite DNA was done in different sheep breeds. The gene frequency, the polymorphism information contents, the number of effective alleles, the heterozygosity, and the genetic distances were studied in four imported meat sheep and Small Tail Han sheep using five microsatellite loci. The crossing effects on the Small Tail Han sheep with four imported meat sheep were tested. The results indicate that there are genetic polymorphisms at five microsatellite loci in five sheep breeds. Five microsatellite loci can be used for genetic diversity evaluation in sheep breeds. The genetic variability of Dorset is the highest, and that of the Small Tail Han sheep is the lowest in the five sheep breeds. The order of heterosis from large to small in four imported meat sheep by the analysis of genetic relationship is White-Suffolk, Black-Suffolk, Dorset, and Texel. This accords with the testing results of actual heterosis. It is feasible to forecast the heterosis of Small Tail Han sheep crossed with imported meat sheep by genetic polymorphism of microsatellite DNA, which will have an important value for sheep breeding in the future.

Microsatellite DNA polymorphisms and the relation with body weight in sea cucumber Apostichopus japonicus

The relationship between microsatellite polymorphism and body weight of captive bred Chinese sea cucumber Apostichopus japonicus was investigated in two local populations in Dalian. Among ten loci discovered, nine show changes except for AJ07 loci. Seven loci were found highly polymorphic in both populations. For each locus in two populations, the average number of alleles is 6.428 6 and 6.285 7, the average observed heterozygosity at 0.225 7 and 0.245 9, the expected heterozygosity at 0.776 8 and 0.748 8, the polymorphism information content (PIC) at 0.709 2 and 0.674 6, respectively. Further analysis show significant correlation between A. japonicus body weight and occurrence markers AJ02 and AJ04. The findings of the relation may be helpful for molecular breeding, as well as the marker-assisted selection of sea cucumbers.

Genetic variation of Chinese and Japanese wild Pacific abalone(Haliotis discus hannai)measured by microsatellite DNA markers

Population differentiation and relationships among three wild populations of the Pacific abalone Haliotis discus hannai collected from coastal seas around China and Japan were estimated using microsatellite DNA analysis. The results obtained with six microsatellite loci showed a high genetic diversity for China and Japan populations. The mean number of alleles per locus ranged from 11.7 to 23.0, and the average of observed and expected heterozygosity ranged from 0. 656 to 0. 721, and from 0. 721 to 0. 793, respectively. The observed genotype frequencies at each locus were mostly in agreement with Hardy - Weinberg expectations with five exceptions. Significant differences were detected between Chinese and Japanese H. discus hannai populations [ Weir and Cocker-ham＇ s fixation index（ Fst ） range ： 0. 020～ 0.023 ; Slatkin＇ s fixation index （Rst） range： 0. 016～ 0. 044 ], and no obvious difference was detected between the samples of Japanese H. discus hannm populations （ Fst = 0. 002 ; Rst = 0. 007）. The level of differentiation among populations is further evidenced by the nNeighbor-joining tree topology on which the Japanese samples were closely clustered, and the Chinese population formed a separate cluster. These results suggest that care should be taken in future management of different populations.

Isolation and Characterization of 17 Microsatellite DNA Loci for Odorrana margaretae(Anura: Ranidae)

We isolated and characterized 17 microsatellite DNA loci for the odorous frog Odorrana margaretae from its transcriptome sequence data. These loci were screened with 24 individuals from Mt. Emei. All loci were polymorphic, with the number of alleles ranging from 2 to 8. The observed and expected heterozygosity, polymorphism information content, ranged from 0.04 to 1, 0.04 to 0.81, and 0.040 to 0.763, respectively. All loci were in linkage equilibrium and six loci were significantly deviated from Hardy-Weinberg equilibrium after sequential Bonferroni corrections. Cross- species amplification test was conducted for ten odorous frog species, and 12 loci were amplifiable in most species. With the high cross-species amplification rates, thesc markers will provide useful molecular tools for conservation genetic and phylogeographic studies on the genus Odorrana and Bamburana.

Preliminary Study on Applicability of Microsatellite DNA Primers from Parasite Protozoa Trypanosoma cruzi in Free-living Protozoa

In this paper, we took the lead in studying on specificity of the microsatellite DNA loci and applicability of mi crosatellite DNA primers in protozoa. In order to study characters of microsatellites in free living protozoa, eight microsatellite loci primers developed from Trypanosoma cruzi (MCLE01, SCLE10, MCLE08, SCLE11, MCLF10, MCLG10, MCL03, MCL05) were employed to amplify microsatellite in four free living protozoa, including Bodo designis, Euglena gracilis FACHB848, Paramecium bruzise and Tetrahymena thermophila BF1. In the amplification systems of P. bruzise, four loci (SCLE10, SCLE11, MCLF10, MCL03) were amplified successfully, and four amplification fragments were in proper size. In genome of E. gracilis FACHB848, five of eight primers brought five clear amplification bands. In B. designis, three (No.4, 5 and 7) of eight loci produced clear and sharp products without stutter bands, whereas no bands appeared in T. thermophila BF1. Further, eight 300-500 bp amplification fragments were cloned and sequenced. Nevertheless, all sequenced products did not contain corresponding microsatellite sequence, although Bodo is in the same order and has the nearest phylogenetic relation with Trypanosoma among these four species. Thus, the microsatellite DNA primers can not be applied among order or more far taxa, and the specificity of microsatellite DNA is very high in protozoa. The results of this study will contribute to our understanding of microsatellite DNA in protozoa.

Prediction of the Best Three-Breed Hybridized Combination of Imported Meat Sheep Using Genetic Polymorphism of Microsatellite DNA

The allelic frequency, the polymorphic information contents （PIC）, the number of effective alleles, the heterozygosity, and the genetic distances were studied in three imported meat sheep （Suffolk, Dorset, Texel） and their F1 crossbred obtained from those crossed with indigenous Small Tail Hun Sheep （Suffolk♂× Small Tail Hun Sheep, SH; Dorset ♂× Small Tail Han Sheep♂, DH; Texel♂× Small Tail Hart Sheep ♀, TH） using six microsatellite DNA loci. The perpormences of three-breed crossbred （Suffolk ♂× DH ♀, Suffolk ♂× TH ♀, Texel ♂× SH ♀, Texel ♂× DH ♀, Dorset ♂× TH ♀, and Dorset ♂× SH ♀ ） were tested. The results indicated that there were genetic polymorphisms at six microsatellite loci in six sheep populations. Six microsatellite loci could be used for genetic diversity evaluation in sheep populations. The order of three-breed heterosis by the analysis of genetic relationship from large to small was Texel ♂× DH ♀, Suffolk ♂× DH ♀, Suffolki ♂× TH ♀, Texel ♂× SH ♀, Dorset ♂×TH ♀, and Dorset ♂× SH ♀, which was in accordance with the testing results on actual heterosis. These results showed that prediction of the best three-breed hybridized combination among sheep breeds by microsatellite DNA polymorphism was feasible, which will have an important value on the reasonable utilization of introduced meat sheep and sheep breeding in our country in the future.

Nuclear and mitochondrial DNA microsatellite instability in hepatocellular carcinoma in Chinese

AIM：To study the nuclear microsatellite instability (nMSI) at BAT26 and mitochondral microsalellite instability (mtMSI) in the occurrence and development of hepatocellular carcinoma and the relationship between nMSI and mtMSI.METHODS: nMSI was observed with PCR and mtMSI with PCR-SSCP in 52 cases of hepatocellular carcinoma.RESULTS:mtMSI was detected in 11 out of the 52 cases of hepatocellular carcinoma (21.2%). Among the 11 cases of hepatocellular carcinoma with mtMSI, 7 occured in one locus and 4 in 2 loci. The frequency of mtMSI in the 52 cases of hepatocellular carcinoma showed no correlation to sex, age,infection of hepatitis B, liver cirrhosis as well as positive AFP of the patients (P>0.05). In addition, nMSI was detected in 3 out of 52 cases of hepatocellular carcinoma (5.8%) and there was no correlation of the incidence of mtMSI to that of nMSI (P>0.05).CONCLUSION:mtMSI may be involved in the coccurrence and development of hepatocellular carcinoma and it is independent of nMSI.

Fabrication of Monodisperse ＂Core-Satellite＂ Nanostructures by DNA-Programming： a Novel Class of Superstructured Building Blocks for Hierarchical Nanoassembly

Monodisperse nanoparticle assembly with tunable structure, composition and properties can be taken as a superstructured building block for the construction of hierarchical nanostruc tures from the bottom up, which also represents a great challenge in nanotechnology. Here we report on a facile and controllable method that enables a high yield fabricatioa of uniform gold nanoparticle （AuNP） coresatellites with definable number （in average） of the satellite particles and tunable coretosatellite distance. The formation of the coresatellite nanostruc tures is driven by programmable DNAbasepairing, with the resulting nanocomplexes being isolatable via gel electrophoresis. By rationally controlling the DNA coverages on the core and shell particles, high production yields are achieved for the assembly/isolation process. As well, benefiting from a minimum DNA coverage on the satellite AuNPs, a strong affinity is observed for the asprepared coresatellites to get adsorbed on proteincoated graphene ox ide, which allows for a twodimensional hierarchical assembly of the coresatellite structures. The resulting hierarchical nanoassemblies are expected to find applications in various areas, including plasmonics, biosensing, and nanocatalysis. The method should be generalizable to make even more complicated and higherorder structures by making use of the structural programmability of DNA molecules.

Isolation and Characterization of 15 Microsatellite DNA Loci for the Alpine Stream Frog Scutiger boulengeri (Anura： Megophryidae)

Fifteen microsatellite DNA markers were developed from a transcriptome assembly of the alpine stream frog （Scutiger boulengeri）.To characterize these loci, we genotyped 23 individuals collected from two sites. Eleven loci were variable, with the number of alleles ranging from one to six within each population. The expected and observed heterozygosities ranged from 0 to 0.78 and from 0 to 0.64, respectively. None of the loci was in linkage disequilibrium and one locus for one population was deviated from will offer useful tools in detecting population structure Tibetan Plateau, where the species resides. the Hardy-Weinberg equilibrium. We hope that these markers of S. boulengeri and in monitoring the fragile ecosystem of the

Morphological and microsatellite DNA diversity of Nigerian indigenous sheep

Background： Sheep is important in the socio-economic lives of people around the world. It is estimated that more than half of our once common livestock breeds are now endangered. Since genetic characterization of Nigerian sheep is still lacking, we analyzed ten morphological traits on 402 animals and 15 microsatellite DNA markers in 384 animals of the 4 Nigerian sheep breeds to better understand genetic diversity for breeding management and germplasm conservation. Results： Morphological traits of Uda and Balami were significantly （P 〈 0.05） higher than Yankasa, which were both higher than West African Dwarf （WAD） sheep. Stepwise discriminant analysis showed tail length, rump height, chest girth, ear length and chest depth as the most discriminating variables for classification. Mahalanobis distances show the least differentiation between Uda and Balami and the largest between WAD and Balami sheep. While 93.3% of WAD sheep were correctly assigned to their source genetic group, 63.9% of Yankasa, 61.2% of Balami and 45.2% of Uda were classified correctly by nearest neighbour discriminant analysis. The overall high Polymorphism Information Content （PIC） of all microsatellite markers ranged from 0.751 to 0.927 supporting their use in genetic characterization. Expected heterozygosity was high for all loci （0.783 to 0.93）. Mean heterozygote deficiency across all populations （0.17] to 0.534） possibly indicate significant inbreeding （P 〈 0.05）. Mean values for FST, FIT and F^s statistics across all loci were 0.088, 0.394 and 0.336 respectively. Yankasa and Balami are the most closely related breeds （DA = 0.184） while WAD and Balami are the farthest apart breeds （DA-- 0.665）, which is coincident with distance based on morphological analysis and population structure assessed by STRUCTURE. Conclusions： These results suggest that within-breed genetic variation in Nigerian sheep is higher than between-breeds and may be a valuable tool for genetic improvement and conservation. The higher genetic variability in Yankasa suggests the presence of unique ancestral alleles reflecting the presence of certain functional genes which may result in better adaptability in characteristics are potentially useful in planning indigenous sheep. more agro-ecological zones of Nigeria. These genetic mprovement and conservation strategies in Nigerian

Genetic Diversity Analysis of Three Guizhou Local Pig Breeds Using Microsatellite DNA Markers

[Objective] The research aimed to provide the genetic basis for the protection, development and utilization of Guizhou local pig breeds. [Method] From 27 pairs of porcine microsatellite primers recommended by Food and Agriculture Organization of the United Nations （FAO） and international Society for Animal Genetics （ISAG）, six pairs （S0155, SW240, IGF1, SW951, SW857, SW24） were selected for microsatellite DNA detection of three Guizhou local pig breeds, including Nuogu Pig, Kele Pig and Guanling Pig. Subsequently, their genetic diversities were analyzed. [ Result] The three pig breeds were high polymorphic at the six microsatellite loci （PIC 〉0.5）. The Nei＇s standard genetic distance of them was 0.206 3 -0.481 5. The genetic distance between Nuogu Pig and Kele Pig Was the closest, and that between Nuogu Pig and Guanling Pig was the furthest. [ Conclusion] The three Guizhou local pig breeds are in high genetic diversities. Nuogu Pig is a special type of Kele Pig, an excellent Chinese local pig breed.

Bioinformatic Analysis of BBTV Satellite DNA in Hainan

Banana bunchy top virus (BBTV),family Nanaviridae,genus Babuvirus,is a single stranded DNA virus (ssDNA) that causes banana bunchy top disease (BBTD) in banana plants.It is the most common and most destructive of all viruses in these plants and is widespread throughout the Asia-Pacific region.In this study we isolated,cloned and sequenced a BBTV sample from Hainan Island,China.The results from sequencing and bioinformatics analysis indicate this isolate represents a satellite DNA component with 12 DNA sequences motifs.We also predicted the physical and chemical properties,structure,signal peptide,phosphorylation,secondary structure,tertiary structure and functional domains of its encoding protein,and compare them with the corresponding quantities in the replication initiation protein of BBTV DNA1.

基于中医证候与精液质量相关参数构建精子DNA碎片预测模型与验证

背景:中医证候与精液质量相关参数相结合,共同预测精子DNA碎片指数(DNA fragmentation index,DFI)异常增高的发生并绘制列线图,能显著提高临床的实操性与应用效能,为临床全面评估精液质量,采取积极干预措施以改善临床结局及制定个体化医疗方案提供依据。目的:探讨基于中医证候与精液质量相关参数构建精子DNA碎片的预测模型与验证。方法:回顾性分析2019年7月至2021年7月在广西壮族自治区南溪山医院中医男科接受中医证候诊断及精子DNA碎片率检查的不育患者共420例,据《人类精液检查与处理实验室手册》(第6版),将其中137例精子DFI>30%患者纳入精子DFI异常增高组,将283例精子DFI≤30%作为对照组;首先采用单因素分析筛选精子DFI异常增高的影响因素,然后采用套索算法(LASSO)校正因子共线性问题并筛选出最佳匹配因子后,将其纳入多因素向前逐步Logistic回归找出其独立影响因素并绘制列线图,最后采用受试者工作曲线、校准曲线、临床决策曲线、临床影响曲线对该预测模型进行区分度与准确度及临床应用效能验证。结果与结论:①单因素分析结果显示,年龄、体质量指数、前向运动率、精子总活率、精子浓度、精子形态学、肾阳虚衰证、湿热下注证、肾精不足证为引发精子DFI异常增高的影响因子(P<0.05);②通过LASSO回归进一步筛选出的最佳匹配因素为年龄、体质量指数、精子总活率、精子浓度、精子形态学、肾阳虚衰证、湿热下注证、肾精不足证(P<0.05);③多因素向前逐步Logistic回归结果显示年龄、体质量指数、精子浓度、精子总活率、湿热下注证、肾阳虚衰证共6项为引发精子DFI异常增高的独立影响因素;④受试者工作曲线显示,模型组曲线下面积为0.760(0.713,0.806),验证组曲线下面积为0.745(0.714,0.776),说明该预测模型具有较好的区分度;⑤校准曲线平均绝对误差0.040,Hosmer-Lemeshow检验P>0.05,表明该模型预测发生精子DFI异常增高的概率与实际发生精子DFI异常增高的概率无显著统计学差异,证实该模型具有较好的准确度;⑥临床决策曲线与临床影响曲线显示,模型组与验证组分别在阈概率值为0.08-0.84与0.09-0.78时具有临床最大净获益,且在该阈概率范围内具有较好的临床应用效能;⑦结果表明,年龄、体质量指数、精子浓度、精子总活率、湿热下注证、肾阳虚衰证为引发精子DFI异常增高的独立影响因素,通过其构建的临床预测模型列线图具有较好的临床预测价值与临床应用效能,可为临床全面评估精液质量、预后与干预及个体化医疗服务提供依据。