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.

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.

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.

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.

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.

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.

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.

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.

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 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.

Microsatellite DNA Variation of the Gametophyte Clones Isolated from Introduced Laminaria japonica (Phaeophyta) and L. longissima of China and Varieties Derived from them

The variation of 90 Laminaria gametophyte clones representing the introduced Laminaria japonica （Group 1） and Laminaria Iongissima （Group 2）, the varieties of L. japonica （Group 3） and the varieties derived from interspecific hybrids （Group 4） was determined with 18 microsatellite markers. The allelic diversity and Nei＇s gene diversity of Group 1 were significantly higher than those of Group 2 （2.9 vs. 1.8 and 0.414 vs. 0.161, respectively）, demonstrating that the variation of the introduced L. japonica is richer than that of L. Iongissima. Both allelic diversity and Nei＇s gene diversity of Group 3 were lower than those of Group 1, indicating that only a portion of variation of L. japonica was incorporated into the varieties of L. japonica. Significant genetic differentiation was detected between four groups and between female （Population 1 ） and male （Population 2） gametophyte clones in each group. The variation among groups accounted for 39.95%, while that among populations accounted for 21.65% of the total. The genetic distance between Group 1 and Group 4 was obviously longer than that between Group 2 and Group 4 （0.686 vs. 0.291）, indicating that maternal gametophyte clone contributed more variation to the hybrids than the paternal gametophyte clone did.

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-

Study on Population Genetic Characteristics of Qinchuan Cows Using Microsatellite Markers

To evaluate the genetic polymorphisms and to search for available molecular markers for Qinchuan cattle, 90 Qinchuan cows were genotyped with 12 microsatellite markers. A total of 247 alleles were detected, with the number of alleles ranging from 13 （INRA005） to 33 （HEL13）, giving a mean number of 21 alleles per locus. The total and mean effective allele number were 142.6229 and 11.8852, respectively. Mean sampling variance of the allele frequency was 2.6036 × 10^-4. Allele size ranges of the 12 microsatellite loci were different. The observed heterozygosity and expected heterozygosity were from 0.7842 （INRA005） to 0.9775 （BM315） and 0.7952 （BM315） to 0.9446 （HEL13）, respectively. Mean observed heterozygosity and mean expected heterozygosity were 0.9117 and 0.9047, respectively. Polymorphism information content values were from 0.7653 （INRA005） to 0.9420 （HEL13）, and mean polymorphism information content of the 12 microsatellite loci was 0.8965. All the 12 microsatellite loci were highly polymorphic, which showed that there were rich genetic polymorphisms at these detected microsatellite loci in Qinchuan cows. At the 12 microsatellite loci, the mean fixation index was -0.0076, reflecting that the degree of heterozygote defect at these loci was not high and deviations from Hardy-Weinberg equilibrium were not significant.

Genetic Analysis of Six Endangered Local Duck Populations in China Based on Microsatellite Markers

Seventeen microsatellite loci were used to assess the genetic variation and population structure of six endangered duck populations in China. PIC, heterozygosity with two estimators of genetic differentiation （FST and GST）, and Nei＇s standard genetic distance were evaluated. The results showed that these six endangered duck populations showed high polymorphism. The propor- tion of inter-population subdivision among the six duck populations ranged between 17.0 and 14.7%. The average heterozygosity was 0.706, 0.702, 0.691, 0.676, 0.604, and 0.660 in the Gaoyou, Liancheng, Jinding, Beijing, Shaoxing, and Jianchang ducks, respectively. The average PIC ranged from 0.561 to 0.663. Finally, the results showed higher genetic diversity and difference above the levels of genetic variation among all the populations.

Isolation via enrichment and characterization of ten polymorphic microsatellite loci in the cuttlefish, Sepiella maindroni de Rochebruns

Ten novel polymorphic microsatellite loci were developed using an enrichment and colony hybridization protocol and characterized for the cuttlefish,Sepiella maindroni.Polymorphism was explored by using 30 individuals from the coastal waters of Zhanjiang,Guangdong province,China,in December 2007.The number of alleles ranged from 5 to 13.The ranges of observed and expected heterozygosity were from 0.578 9 to 1.000 0 and 0.682 8 to 0.925 7,respectively,and the average polymorphic information content （P IC） was 0.778 5.These microsatellite loci will certainly facilitate the detection of the genetic variation and population structure of S.maindroni.

Blast-Resistance Inheritance of Space-Induced Rice Lines and Their Genomic Polymorphism by Microsatellite Markers

To understand the resistance inheritance basis of space-induced rice lines to blast, and to probe mutants＇ genomic DNA polymorphism compared with ground control by microsatellite markers, three space-induced lines were crossed with a highly susceptible variety LTH, and their F1 and F2 populations were inoculated by two representative blast isolates with broad pathogenicity to analyze their resistance inheritance basis. Meanwhile three mutant lines and the ground control were analyzed by 225 rice SSR （simple sequence repeat） primer pairs selected throughout the 12 chromosomes of whole rice genome, to scan the mutagenesis in genome of the mutant lines. The results indicated the blast-resistant genes harbored in these mutant lines were dominant. It was demonstrated that the resistance of mutant H1 to isolate GD0193 and GD3286 was controlled by a single gene, respectively; while mutants H2 and H3 were controlled by two pairs of major genes against isolate GD3286 and H2 showed complicated genetic mechanism to isolate GD0193. H3＇s resistance to isolate GD0193 was verified to be controlled by a single gene. According to the results of SSR analysis, three mutant lines showed different mutant rates as compared with the ground control, and the mutant rates also varied. Resistance genes can be induced from rice by space mutation, and different genomic variations were detected in blast-resistant lines.

Microsatellite-Based Genetic Differentiation and Phylogeny of Sheep Breeds in Mongolia Sheep Group of China

In the present study we studied the genetic structure of five Chinese sheep populations of Hu sheep, Tong sheep, Small-tailed Han sheep, Tan sheep, and Wadi （WD） sheep using 15 microsatellite loci. The results showed that the FIT, FST, and FIS statistics computed for the complete dataset had the following values, 0.523±0.140, 0.363±0.131 and 0.263±0.092, respectively. All loci were significantly contributed to the genetic differentiation among population （P0.001）. There is no relationship between the scatter of pairwise FST geographical distance points as geographical distance increases between the five populations. Membership probabilities and genetic structure of sheep populations were estimated when K=2, the populations were classified into Hu, Tong, Han and WD, and Tan sheep group. However, when K=3, the populations were classified into Hu and Tong, Han and WD, and Tan sheep group. The findings supported the previous literatures that these populations are originated on different time stages from the primogenitor population and communicated genetically with each other by natural and artificial selection in different ecological environment.

Profile of candidate microsatellite markers in Sebastiscus marmoratus using 454 pyrosequencing

Sebastiscus marmoratus is an important sedentary ovoviparous fish distributed in near-shore coastal waters from the coast of China to Japan. Candidate S. marmoratus microsatellite markers were developed in the present study using 454 pyrosequencing, and the marker profile was analyzed. A total of 2 000 000 raw sequence reads were assembled to reduce redundancy. Among them, 1 043 dinucleotide, 925 trinucleotide, 692 tetranucleotide, and 315 pentanucleotide repeats were detected. AC repeats were the most frequent motifs among the dinucleotide repeats, and AAT was the most abundant among the trinucleotide repeats. AAAT, ATAG, and ATCC were the three most common tetranucleotide motifs, and AAGAT and AATAT were the most dominant pentanucleotide motifs. The greatest numbers of loci and potentially amplifiable loci were found in dinucleotide repeats, whereas trinucleotide repeats had the fewest. In summary, a wide range of candidate microsatellite markers were identified in the present study using a rapid and efficient 454 pyrosequencing approach.

Correlation of Simple Sequence Repeat (SSR) Variants in the Leader Sequence of the waxy Gene with Amylose Content of the Grain in Rice

Variation of two simple sequence repeats (SSRs) in the leader region of the waxy gene was analyzed in a sample of 74 accessions, including Oryza sativa L. ssp. indica, japonica and wild rice (O.rufipogon) representing a wide distribution range of amylose content (AC) in cultivated rice. Eight alleles were detected in the (CT) n motif and two alleles were resolved in the (AATT) n motif. The distribution of the alleles of the two SSRs was quite uneven as detected by the (CT) n motif. The repeat numbers of the two SSR motifs, (CT) n and (AATT) n, appeared to be inversely related such that the total length of this region was maintained. AC of the varieties was highly correlated with the length of SSRs. Differences in AC among the various SSR genotypes were statistically highly significant as analyzed using genotypes of both SSR motifs. Although the SSR variation did not seem to have obvious function in the synthesis of the starch synthase encoded by the waxy gene, the almost perfect correlation between the two SSRs and AC level could be used for quality improvement in rice breeding programs.