Discordant patterns of genetic variation between mitochondrial and microsatellite markers in Acanthogobius ommaturus across the coastal areas of China

Acanthogobius ommaturus, which belongs to the family Gobiidae, is a euryhaline and demersal fish that is widely distributed in the coastal areas, harbors, and estuaries of China, D. P. R. Korea and Japan. In this study, the genetic diversity and genetic structure of five geographical populations of A. ommaturus was assessed using the mitochondrial hypervariable region gene and microsatellite markers. The results of the two genetic markers indicated that the A. ommaturus populations had a high level of genetic diversity. The mitochondrial marker detected weak genetic differentiation among populations, and the Neighbor-Joining tree showed that there was no obvious pedigree branches and geographic structure as well. However, population of Zhoushan showed significant genetic differentiation with other populations by microsatellite markers. The population of A.ommaturus has not experienced bottleneck effect recently. We speculated that the Pleistocene climate change and juvenile fish dispersal played an important role in the population differentiation of A. ommaturus.

Development and Characterization of Microsatellite Markers for Harpadon nehereus Based on High-Throughput Sequencing and Cross-Species Amplification in Three Myctophiformes Fishes

Harpadon nehereus is a widespread economical fish found in the coastal seas of China and has important ecological value in the marine ecosystem.H_(o)wever,its germplasm resources have been seriously degraded due to natural factors and anthropogenic activities.In this study,high-throughput sequencing was applied to search for microsatellite loci in H.nehereus transcriptome to provide references for its resource conservation and utilization.Polymorphic loci were developed by non-denaturing polyacrylamide gel electrophoresis,and their cross-species amplified ability was detected in three related species.A total of 5652 microsatellites were identified from 16974320 unigenes.Among the primer pairs designed for 100 SSRs for PCR amplification,80%were successfully amplified,and 26 loci were polymorphic with a high number of alleles from 3 to 11 each.The expected(H_(e))and observed(H_(o))heterozygosities were 0.355–0.885 and 0.375–0.958,respectively.Most of the loci were highly polymorphic(polymorphism information content:0.316–0.852;mean:0.713),and these markers can be applied in the population genetic diversity research of H.nehereus.H_(o)wever,the transferability of these primers was low,probably because of the close relation of the collected species.In follow-up work,simple sequence repeats will be excavated with genome-based technologies,and related species will be gathered to address the present inadequacies.

Assessment of Genetic Variability and Inter-specific Relationships among Four Species of Groupers (Genus Epinephelus) from South China Sea Using Microsatellite Markers

[Objective] The study aimed to evaluate the genetic variability and interspecific relationships among four species of groupers from South China Sea, including E. fario, E. merra, E. malabaricus and E. coioides. [Method] Twenty one mircosatellite loci of groupers were selected from GenBank and eight high polymorphic loci were used to further genetic analysis. [Result] The mean number of alleles per locus （A）, effective number of alleles （Ne）, mean polymorphism information content （PIC）, observed heterozygosity （Ho） and expected heterozygosity （He） were 4.38±1.60, 3.69±0.86, 0.69±0.08, 0.67±0.08, 0.72±0.06 in E. malabaricus; 3.88±1.13, 3.55±1.04, 0.66±0.10, 0.68±0.21, 0.70±0.08 in E.coioides; 6.00±1.07, 4.68±0.65, 0.78±0.03, 0.73±0.25, 0.79±0.03 in E. fario; 5.50±1.07, 4.58±0.80, 0.76±0.05, 0.75±0.18, 0.78±0.04 in E. merra, respectively. [Conclusion] We compared the values above, the order of the genetic variability among these grouper species was E. fario E. merra E. malabaricus E. coioides. We found that the level of genetic variability of these groupers species was relatively higher than that of other marine fish, so their genetic status was good. In addition, the analyisis of genetic relationship showed that E. malabaricus and E. coioides was the closest and it was the farthest between E. merra and E. coioides.

Genetic Analysis and Linkage Mapping in a Resource Pig Population Using Microsatellite Markers

The use of markers and linkage map construction are important for QTL mapping in pigs. In this article, the genetic characteristics were studied and the linkage map was constructed in a pig resource population including 214 individuals by typing 39 microsatellite marker loci on Sus scrofa chromosomes, SSC4, SSC6, SSC7, SSC8, and SSC13. Results indicated that the average allele number, the average observed beterozygosity （Ho）, and the average polymorphism information content （PIC） in F1 and F2 population were 3.2, 0.528, 0.463 and 3.2, 0.496, 0.447, respectively. In the pig resource population, the average informative meiosis （IM） was 217.4 （44-316）, and the average linkage map length between the two sexes on the five chromosomes were 172.3 cM （SSC4）, 168.7 cM （SSC6）, 191.7 cM （SSC7）, 197.3 cM （SSC8）, and 178.3 cM （SSC13）. The orders of microsatellite marker loci in the linkage maps were identical to, but the length was greater than, those of USDA-MARC reference map. The results of this research showed the genetic relationship and genetic characteristics of the microsatellite markers in the pig resource family population, and the linkage map could be used to for QTL mapping in the subsequent study.

Identification of seeds of Pinus species by microsatellite markers

The 276 pair-primers （nuclear and chloroplast microsatellite） developed from seven species of Pinaceae were selected and identified for cross-species transferability to ten Pinus species （P massoniana, P kesiya, P tabulaeformis P densiflora, P thunbergii, P caribaea, P taeda, P yunnanensis, P densata, P sylvestris）belonging to Sect. Pinus by BSA （bulked segregate analysis） method. The results showed that 23 of 276 （8.0%） markers were successful to have amplification product in ten species, and 5 of 23 （21.7%） were polymorphic cross species and lack of polymorphic within species. Eight of 10 Pinus species were identified by using single primer, two and more combination of primers, but there were still no effective SSR primers for identifying other 2 species （P. kesiya and P. densata）.

Screening microsatellite markers from EST sequences of bay scallop Argopecten irradians

Seventy-five simple sequence repeats （SSRs） were identified by the bioinformatic analysis from 5008 expressed sequence tags （ESTs） of Argopecten irradians. Among the SSRs, the number of repeat nucleotide varied from 2 to 6. Dinucleotide and trinueleotide repeat motifs were dominant in EST-SSRs of bay scallop, with a proportion of 80% over the total screened SSRs. Twenty-nine pairs of primer were designed based on the flank sequences of the selected ESTs using the software of Primet 5, and verified under the given PCR reaction condition. Eighteen of the 29 primer pairs resulted in the expected products, while the remaining either failed to produce any fragments or yielded products over expected size. Thirteen of the 18 SSRs, accounting for 72%, were detected to show polymorphism in the examined scallop samples. A preliminary test in this study indicated that the majority of the identified SSRs were informative in the cultured bay scallops, making them suitable for the population and other genetic analysis. EST-SSR markers have more advantages than the traditional genomic-derived SSRs and there is a wide range of application in comparative mapping, functional gene cloning and marker assisted selection. This research provides a reference to the identification of EST-SSRs with relative bioinformatic analysis from aquaculture species, as well as to those with a large number of ESTs.

Mapping QTLs Affecting Economic Traits on BTA3 in Chinese Holstein with Microsatellite Markers

It had been demonstrated that the strong and highly significant quantitative trait locus（QTL） can affect protein percentage on Bos Taurus Autosome 3（BTA3） at the position 52 cM, near the microsatellite DIK4353, with the 95% confidence interval spanning from 25 to 57 cM in Chinese Holstein population using QTL-express, MQREML, and GRIDQTL softwares. This study herein focused on such region of fine mapping QTLs for milk production and functional traits with 16 microsatellite markers with coverage of 33 cM between the markers BMS2904 and MB099 on BTA3 in a daughter-designed Chinese Holstein population. A total of 1 298 Holstein cows and 7 sires were genotyped for 16 microsatellites with ABI 3700 DNA sequencer. The variance components QTL linkage analysis（LA） and linkage-disequilibrium（LD） analysis（LA/LD） was performed to map QTLs for 7 traits, i.e., 305-d milk yield, fat yield, protein yield, fat percentage, protein percentage, somatic cell score and persistency of milk yield. Four strong and highly significant QTLs were detected for fat yield, fat percentage, protein percentage and somatic cell score at the position 40, 30, 27 and 26 cM, respectively. Two minor QTLs for milk yield and persistency of milk yield were identified at 42 and 46 cM, respectively. These findings provided a general idea for the fine mapping of the causal mutation for milk production and functional traits on BTA3 in the future.

Development and characterization of novel polymorphic microsatellite markers for the Korean freshwater snail Semisulcospira coreana and cross-species amplification using next-generation sequencing

Korean freshwater snails of the genus Semisulcospira are widely distributed across East Asia.It has been a very popular nutritional food in Korea,and is an ecologically important water quality indicator because it lives only in clean water.However,no microsatellite markers have been generated to study the population genetic diversity of this genus.In the present study,we developed and characterized 18 novel microsatellite loci from Semisulcospira coreana genomic DNA.The microsatellites were isolated using 454 GS-FLX titanium sequencing and 18 markers were used for genotyping in S.coreana.In addition,we also tested the cross-species transferability of the microsatellite markers in four additional Semisulcospira spp.We identified 18 polymorphic loci and the number of alleles per loci,and their polymorphism information content values ranged from 2 to 17 and 0.203 to 0.902,respectively.The observed and expected heterozygosities of the loci ranged from 0.063 to 0.924 and 0.226 to 0.924,respectively.According to the analysis of the cross-species transferability of these markers,four species,S.forticosta,S.gottschei,S.tegulata,and S.libertina,showed a very high transferability(80%–85%).These results show that this set of nuclear markers could be useful for population genetics studies of this species and closely related species.

Inbreeding and genetic diversity analysis in a hatchery release population and clones of Rhopilema esculentum based on microsatellite markers

Ten microsatellite markers were used to analyze the levels of genetic diversity and inbreeding in a hatchery release population of Rhopilema esculentum Kishinouye （Scypbozoa： Rhizostomatidae）. A total of 85 alleles were detected in 600 individuals. Within-population levels of observed （Ho） and expected （He） heterozygosity ranged from 0.152 to 0.839 （mean=0.464） and from 0.235 to 0.821 （mean=0.618）, respectively. The polymorphism information content （PIC） of each marker ranged from 0.207 to 0.795 with an average of 0.580, indicating that the hatchery population maintained a high level of genetic diversity. Inbreeding levels were estimated in the hatchery population and the inbreeding coefficient was 0.203. This result revealed that a certain level of inbreeding occurred within the population. Meanwhile, we also determined genetic diversity at the clone level. Several polyps from the same scyphistomae were genotyped at the ten microsatellite loci and there was virtually no difference in their genotypes. Furthermore, we calculated the probabilities of exclusion. When both parents were known, the average exclusion probability often loci was 99.99%. Our data suggest that the ten microsatellite markers can not only be used to analyze the identity of individuals but they can also be applied to parentage identification. Our research provides a theoretical basis and technical support for genetic diversity detection and reasonable selection of R. esculentum hatchery populations. These findings support the use of releasing studies and conservation of R. esculentum germplasm resources.

Mapping of a Major Stripe Rust Resistance Gene in Chinese Native Wheat Variety Chike Using Microsatellite Markers

Chike （accession number Su1900）, a Chinese native wheat （Triticum aestivum L.） variety, is resistant to the currently prevailing physiological races of Puccinia striiformis Westend. f. sp. tritici in China. Genetic analysis indicated that resistance to the physiological race CY32 of the pathogen in the variety was controlled by one dominant gene. In this study, BSA （bulked segregant analysis） methods and SSRs （simple sequence repeats） marker polymorphic analysis are used to map the gene. The resistant and susceptible DNA bulks were prepared from the segregating F2 population of the cross between Taichung 29, a susceptible variety as maternal parent, and Chike as paternal parent. Over 400 SSR primers were screened, and five SSR markers Xwmc44, Xgwm259, Xwmc367, Xcfa2292, and Xbarc80 on the chromosome arm 1BL were found to be polymorphic between the resistant and the susceptible DNA bulks as well as their parents. Genetic linkage was tested on segregating F2 population with 200 plants, including 140 resistant and 60 susceptible plants. All the five SSR markers were linked to the stripe rust resistance gene in Chike. The genetic distances for the markers Xwmc44, Xgwm259, Xwmc367, Xcfa2292, and Xbarc80 to the target gene were 8.3 cM, 9.1 cM, 17.2 cM, 20.6 cM, and 31.6 cM, respectively. Analysis using 21 nulli-tetrasomic Chinese Spring lines further confirmed that all the five markers were located on chromosome lB. On the basis of the above results, it is reasonable to assume that the major stripe rust resistance gene YrChk in Chike was located on the chromosome arm 1BL, and its comparison with the other stripe rust resistance genes located on 1B suggested that YrChk may be a novel gene that provides the resistance against stripe rust in Chike. Exploration and utilization of resources of disease resistance genes in native wheat varieties will be helpful both to diversify the resistance genes and to amend the situation of resistance gene simplification in the commercial wheat cultivars in China.

Intra-breed Genetic Variation of Fragrance Pigs Detected by Microsatellite Markers

[Objective] The genetic background of four types of fragrance pigs was studied using microsatellite molecular markers,in order to fully understand the genetic resources of miniature pigs in China.[Method] Using 27 pairs of microsatellite loci jointly recommended by Food and Agricultural Organization（FAO）and International Society for Animal Genetics（ISAG）,we detected the genotypes of 200 fragrance individuals belonging to four types（Jiuyang fragrance pig,Jianbai fragrance pig,Congjiang fragrance pig and Huanjiang fragrance pig）,and analyzed their Inter-and intra-breed genetic variations.[Result]The 23 loci detected in the test were high polymorphic;the mean heterozygosity（H） of Jiuyang fragrance pig,Jianbai fragrance pig,Congjiang fragrance pig and Huanjiang fragrance pig were 0.683 6,0.667 9,0.697 3 and 0.702 2,and their mean polymorphism information contents（PIC） were 0.6263,0.6063,0.6420 and 0.6415,respectively.[Conclusion]Four types of fragrance pigs detected in the test all had high intra-breed genetic variability.

Development of microsatellite markers and their utilization in genetic diversity analysis of cultivated and wild populations of the mud carp (Cirrhina molitorella)

Microsatellite markers have been increasingly used in genetic studies on fishery species because of their high applicability in selective breeding programs. Here we reported the development of microsatellite markers and their utilization in mud carp （Cirrhina molitorella）. An （CA）15 enriched library has been constructed for mud carp, using the magnetic beads enrichment procedure. Sequence analysis of 60 randomly picked positive colonies indicate that 56 （93.3%） of the colonies contain microsatellites. Microsatellite polymorphism was assessed using 10 mud carp individuals, and 12 microsatellite loci turned out to be polymorphic. We utilized these loci to study the genetic diversity of a wild population （WM） and a cultured population （CM） of the mud carp. A total of 109 alleles were detected with an average of 9.08 alleles per locus. The mean value of the observed heterozygosity of WM and CM was 0.6361 and 0.6417, respectively, and significant decrease of genetic diversity in CM was not observed. The genetic distance between the two populations was 0.1546 and the value of Gsr was 0.0473. This showed that there existed a slight genetic differentiation between WM and CM.

Development and Characterization of Microsatellite Markers in Brassica rapa ssp.chinensis and Transferability Among Related Species

Simple sequence repeat （SSR） or microsatellite marker is a valuable tool for several purposes, such as mapping, fingerprinting, and breeding. In the present study, an intersimple sequence repeat （ISSR）-PCR technique was applied for developing SSR markers in non-heading Chinese cabbage （Brassica rapa）. A total of 190 SSRs were obtained. Among these, AG or CT （54.7%） was the most frequent repeat, followed by AC or GT （31.6%） of the microsatellites. The average number of the SSRs length array was 16 and 10 times, respectively. Based on the determined SSR sequences, 143 SSR primer pairs were designed to evaluate their transferabilities among the related species of Brassica. The number of alleles produced per marker averaged 2.91, and the polymorphism information content （PIC） value ranged from 0 to 0.863 with an average of 0.540. Monomorphism was observed in 16 primer pairs. The transferability percentage in CC genome was higher than in BB genome. More loci occurred in the BBCC genome. This result supported the hypothesis that BB genome was divergent from A and C genomes, and AA and CC genomes were relatively close. The polymorphic primers can be exploited for further evolution, fingerprinting, and variety identification.

Screening and application of microsatellite markers for genetic diversity analysis of Oriental White Stork (Ciconia boyciana)

The Oriental White Stork (Ciconia boyciana) is a large and endangered waterbird in East Asia.Research on conservation genetics of this species is urgently needed.In this study,microsatellite marking technology was used for screening and analysis of genetic diversity of microsatellite markers in Oriental White Storks.A total of 36 pairs of microsatellite primers were used,of which 7 pairs came from Ciconia ciconia,12 pairs from Nipponia nippon and 17 pairs from Ardea herodias.Microsatellite loci were screened from 23 individuals of the Oriental White Stork and 11 microsatellite loci were found with high polymorphism.The number of population alleles ranged from 3 to 11,averaging 7.09.The average expected heterozygosity (H e) was 0.7816 and the average polymorphism information content (PIC) 0.7172,suggesting a relatively high genetic diversity in the population.

Germplasm of breeding Pseudosciaena crocea as revealed by microsatellite markers

The germplasm of breeding large yellow croaker （Pseudosciaena crocea Richardson） was revealed using 12 microsatellite markers. The results showed that the genetic diversities were on a mediated level in the bred Daiqu and Min - Yue stocks and two hybrid groups, as represented by 4.83 of the mean number of alleles and 0.561 of the average observed heterozygosity. The value of pair- wise differentiation coefficient（ FST ） was only 13. 1% between Daiqu and Min -Yue stocks, demonstrating the low level of differentiation and a close relationship. However, STRUCTURE simulations and phylogenetic tree based on the UPGMA method supported that they are geographically different populations of the same species with distinct genetic structures. Examinations of individual admixture showed that Min - Yue stock had been contaminated by alien individuals. Moreover, the genetic structures of the two hybrid groups resembled those of their parents, especially affected more by their female parents. Finally, the values of average observed heterozygosity between parents and their ascendants were compared and tested, as a result of no detectable differences （P 〉 0. 05）.

Genetic variability of half-smooth tongue sole Cynoglossus semilaevis populations using microsatellite markers

Ten highly variable microsatellite loci were performed in order to evaluate the genetic variability of wild and hatchery samples of half-smooth tongue sole Cynoglossu＇s semilaevis. A group of 200 genotypes belonging to four wild samples, Laizhou （LZ）, Weihai （WH）, Qingdao （QD）, Rizhao （RZ） and one hatchery sample, Mingbo （MB）, were screened. All of the： ten microsatellite loci screened in this study showed marked polymorphism. A total of 70 different alleles were observed over all loci. The number of alleles per locus ranged from 4.2 to 1006 the average of expected and observed heterozygosity ranged from 0.67 to 0.82, and from 0：77 to0.87 respectively. A total of 10, 16, 10, 11 and 5 unique alleles each were found in LZ, WH~ QD：RZ and MB populations. The effective number of alleles varied from 2.87 for HSTS_7 to 6.83 for HSTS_h. The number of average genotypes ranged from 6.0 for HSTS a to 15.4 for HSTS_h： As compared with the wild populations, the hatchery population, MB, showed significant genetic changes such as fewer alleles per locus （P〈0.05）, a smaller number of low frequency alleles （P〈0.05）, a small number of unique alleles and a small number of genotypes （P〈0.05）, all indicative of a reduction in genetic diversity.

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.

Morphometric analysis and fluorescent microsatellite markers to evaluate the genetic diversity of five populations of Penaeus japonicus in China

To understand the current status of germplasm resources of Penaeus japonicus,in this study,genetic diversity was analyzed based on morphometric analysis and fluorescent microsatellite markers.For morphometric analysis,the cumulative ratios of the PC1 and PC2(85.90%)were greater than 75%,which can explain the morphological variation among populations.For microsatellite analysis,the allele number in 10 microsatellite loci ranged from 27 to 53,and the effective allele number ranged from 21 to 42.The observed heterozygosity ranged from 0.463 to 0.983,whereas expected heterozygosity ranged from 0.932 to 0.966,and the average polymorphism information content was 0.947,which indicated a high level of genetic diversity.The unbiased genetic distance among populations ranged from 0.230 to 0.431,and the genetic similarity coefficient ranged from 0.654 to 0.782.Analysis of molecular variance(AMOVA)showed that the genetic variation within individuals was the main source of variation,accounting for 88.55%,and only 1.49%of the genetic variation came from among populations.

Novel microsatellite markers suggest the mechanism of parthenogenesis in Extatosoma tiaratum is automixis with terminal fusion

Parthenogenetic reproduction is taxonomically widespread and occurs through various cytological mechanisms, which have different impact on the genetic variation of the offspring. Extatosoma tiaratum is a facultatively parthenogenetic Australian insect （Phasmatodea）, in which females oviposit continuously throughout their adult lifespan irre- spective of mating. Fertilized eggs produce sons and daughters through sexual reproduction and unfertilized eggs produce female offspring via parthenogenesis. Here, we developed novel microsatellite markers for E. tiaratum and characterized them by genotyping indi- viduals from a natural population. We then used the microsatellite markers to infer the cytological mechanism of parthenogenesis in this species. We found evidence suggesting parthenogenesis in E. tiaratum occurs through automixis with terminal fusion, resulting in substantial loss of microsatellite heterozygosity in the offspring. Loss of microsatel- lite heterozygosity may be associated with loss of heterozygosity in fitness related loci. The mechanism of parthenogenetic reproduction can therefore affect fitness outcomes and needs to be considered when comparing costs and benefits of sex versus parthenogenesis.

Parentage assignment and parental contribution analysis in large yellow croaker Larimichthys crocea using microsatellite markers

The large yellow croaker Larimichthys crocea is one of the most important fish species in China. To estimate the re- productive success of breeders, three independent full-factorial crosses were created and the fins of breeders and progenies were sampled for microsatellite analysis. Out of 959 offspring from three sets, 99.6% were assigned to their parents using 6-7 mi- crosatellite markers. In all crosses, some parent pairs produced a large number of offspring and some parent pairs did not produce any offspring. The contributions of male or female parents were unequal, ranging from 1.0-89.3% across the three sets. The loss of putative Ne was 69.6% in set 1, 31.2% in set 2 and 57.6% in set 3. These results suggest that the unequal contribution of parents is universal in artificial breeding ofL. crocea, especially in a small population, and this should be taken into account in hatcheries or when releasing animals for resource enhancement