Application of immune checkpoint inhibitors and microsatellite instability in gastric cancer

In this editorial we comment on the article by Li published in the recent issue of the World Journal of Gastroenterology.We focus specifically on the application of immune checkpoint inhibitors(ICIs)and microsatellite instability(MSI)in gastric cancer(GC).The four pillars of GC management have long been considered,including surgery,chemotherapy,radiotherapy and targeted therapy.However,immunotherapy has recently emerged as a“fifth pillar”,and its use is rapidly expanding.There are four principal strategies for tumor immunotherapy:ICIs,tumor vaccines,adoptive immunotherapy and nonspecific immunomodulators.Of them,ICIs are the most advanced and widespread type of cancer immunotherapy for GC.Recent breakthrough results for ICIs have paved the way to a new era of cancer immunotherapy.In particular,inhibition of the PD-1/PD-L1 axis with ICIs,including nivolumab and pembrolizumab,has emerged as a novel treatment strategy for advanced GC.Unfortunately,these therapies are sometimes associated with often subtle,potentially fatal immune-related adverse events(irAEs),including dermatitis,diarrhea,colitis,endocrinopathy,hepatotoxicity,neuropathy and pneumonitis.We must be aware of these irAEs and improve the detection of these processes to prevent inappropriate discharges,emergency department revisits,and downstream complications.Recent studies have revealed that MSI-high or mismatch-repair-deficient tumors,regardless of their primary site,have a promising response to ICIs.So,it is important to detect MSI before applying ICIs for treatment of GC.

Tislelizumab in previously treated,locally advanced unresectable/metastatic microsatellite instability-high/mismatch repair-deficient solid tumors

Objective:The open-label,phase II RATIONALE-209 study evaluated tislelizumab(anti-programmed cell death protein 1 antibody)as a tissue-agnostic monotherapy for microsatellite instability-high(MSI-H)/mismatch repair-deficient(dMMR)tumors.Methods:Adults with previously treated,locally advanced unresectable or metastatic MSI-H/dMMR solid tumors were enrolled.Patients received tislelizumab 200 mg intravenously every 3 weeks.Objective response rate(ORR;primary endpoint),duration of response(DoR),and progression-free survival(PFS)were assessed by independent review committee(Response Evaluation Criteria in Solid Tumors v1.1).Results:Eighty patients were enrolled and treated;75(93.8%)patients had measurable disease at baseline.Most had metastatic disease and received at least one prior therapy for advanced/metastatic disease(n=79;98.8%).At primary analysis(data cutoff July 8,2021;median follow-up 15.2 months),overall ORR[46.7%;95%confidence interval(95%CI),35.1−58.6;one-sided P<0.0001]and ORR across tumor-specific subgroups[colorectal(n=46):39.1%(95%CI,25.1–54.6);gastric/gastroesophageal junction(n=9):55.6%(95%CI,21.2−86.3);others(n=20):60.0%(95%CI,36.1−80.9)]were significantly greater with tislelizumab vs.a prespecified historical control ORR of 10%;five(6.7%)patients had complete responses.Median DoR,PFS,and overall survival were not reached with long-term follow-up(data cutoff December 5,2022;median follow-up 28.9 months).Tislelizumab was well tolerated with no unexpected safety signals.Treatment-related adverse events(TRAEs)of grade≥3 occurred in 53.8%of patients;7.5%of patients discontinued treatment due to TRAEs.Conclusions:Tislelizumab demonstrated a significant ORR improvement in patients with previously treated,locally advanced unresectable or metastatic MSI-H/dMMR tumors and was generally well tolerated.

Comparative effectiveness of immunotherapy and chemotherapy in patients with metastatic colorectal cancer stratified by microsatellite instability status

BACKGROUND Immunotherapy have demonstrated promising outcomes in patients with high microsatellite instability(MSI)(MSI-H)metastatic colorectal cancer.However,the comparative effectiveness of Immunotherapy and chemotherapy for patients with low MSI(MSI-L),and microsatellite stable(MSS)metastatic colorectal cancer remains unclear.AIM To investigate immunotherapy vs chemotherapy for treatment of MSI-L/MSS metastatic colorectal cancer,and to evaluate the success of immunotherapy against chemotherapy in managing MSI-H metastatic colorectal cancer during a follow-up of 50 months.METHODS We conducted a retrospective cohort study using the National Cancer Database(NCDB)to evaluate the overall survival(OS)of patients with metastatic colorectal cancer treated with immunotherapy or chemotherapy.The study population was stratified by MSI status(MSI-H,MSI-L,and MSS).Multivariable Cox proportional hazard models were used to assess the association between treatment modality and OS,adjusting for potential confounders.RESULTS A total of 21951 patients with metastatic colorectal cancer were included in the analysis,of which 2358 were MSI-H,and 19593 were MSI-L/MSS.In the MSI-H cohort,immunotherapy treatment(n=142)was associated with a significantly improved median OS compared to chemotherapy(n=860).After adjusting for potential confounders,immunotherapy treatment remained significantly associated with better OS in the MSI-H cohort[adjusted hazard ratio(aHR):0.57,95%confidence interval(95%CI):0.43-0.77,P<0.001].In the MSS cohort,no significant difference in median OS was observed between immunotherapy treatment and chemotherapy(aHR:0.94,95%CI:0.69-1.29,P=0.715).CONCLUSION In this population-based study using the NCDB,immunotherapy treatment was associated with significantly improved OS compared to chemotherapy in patients with MSI-H metastatic colorectal cancer,but not in those with MSI-L/MSS metastatic colorectal cancer.Further studies are warranted to determine the optimal therapeutic approach for patients with MSI-L/MSS metastatic colorectal cancer.

Simulation of a soft-gamma-ray polarimeter on board a microsatellite

Gamma-ray polarimetry is a new and prospective tool for studying extremely high-energy celestial objects and is of great significance for the field of astrophysics.With the rapid development of microsatellite technology,the advantages of space exploration have become increasingly apparent.Therefore,we simulated a soft-gamma-ray polarimeter for a microsatellite based on the Compton scattering principle.We performed detailed Monte Carlo simulations using monoenergetic gamma-ray linear-polarization sources and Crab-like sources in the energy range of 0.1-10 MeV considering the orbital background.The polarimeter exhibited excellent polarization detection performance.The modulation factor was 0.80±0.01,and the polarization angles were accurate within an error of 0.2°at 200 keV for on-axis incidence.For the Crab-like sources for on-axis incidence,the polarization degrees were consistent with the set values within the error tolerance,the modulation factor was 0.76±0.01,and the minimum detectable polarization reached 2.4%at 3σfor an observation time of10^(6) s.Additionally,the polarimeter exhibited recoil electron tracking,imaging,and powerful background suppression in a large field of view(FoV;∼2πsr).The proposed polarimeter meets the requirements of a space soft-gamma-ray polarization detector and has promising research prospects.

Modular flexible“Tetris”microsatellite platform based on sandwich assembly mode

In this paper,a flexible modular“Tetris”microsatellite platform is studied to implement the rapid integration and assembly of microsatellites.The proposed microsatellite platform is fulfilled based on a sandwich assembly mode which consists of the isomorphic module structure and the standard mechanical-electric-data-thermal interfaces.The advantages of the sandwich assembly mode include flexible reconfiguration and efficient assembly.The prototype of the sandwich assembly mode is built for verifying the performance and the feasibility of the proposed mechanical-electric-data-thermal interfaces.Finally,an assembly case is accomplished to demonstrate the validity and advantages of the proposed“Tetris”microsatellite platform.

Bat 26 Microsatellite Instability in Oral Cavity Cancers in Senegal

Oral cavity cancers are part of head and neck cancers. They have become frequent in the world in general and Senegal in particular. This study evaluates microsatellite instability tumors in oral cavity cancers in Senegal. Forty cancerous tissues, 20 healthy tissues, and 12 blood tissues were included in this study. These tissues were collected from each patient during the biopsy after obtaining consent. DNA extraction, Polymerase Chain Reaction (PCR) and sequencing were carried out to obtain sequences. Mutation surveyor, Bioedit and Dnasp software were used to perform our analyses. High instability was found in 57.5% of patients with cancer. Moreover, 90% of the patients had the same motif on healthy and cancerous tissue. Furthermore, 26.12%, 20.72%, and 11.71% polymorphic sites were found in cancerous, healthy and blood tissue respectively. Thus, a similarity between cancerous and healthy tissues seems to exist. This implies that instability of the Bat 26 microsatellite could occur early in the occurrence of oral cavity cancers.

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.

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.

Discrepancy among microsatellite instability detection methodologies in non-colorectal cancer:Report of 3 cases

BACKGROUND Microsatellite instability(MSI)is a predictive biomarker for cancer immunotherapy.The tumor-agnostic nature of MSI makes it a denominator for immunotherapy in several solid tumors.It can be assessed using next-generation sequencing(NGS),fluorescent multiplex PCR,and immunohistochemistry(IHC).CASE SUMMARY Here,we report 3 cases with discordant MSI results detected using different methods.A cholangiocellular carcinoma case revealed proficient mismatch repair(MMR)by IHC but high MSI(MSI-H)by liquid NGS.A cervical cancer case revealed deficient MMR by IHC,microsatellite stable by PCR,and MSI-H by NGS.Lastly,an endometrial cancer case revealed proficient MMR by IHC but MSI-H by NGS.CONCLUSION IHC for MMR status is the first choice due to several advantages.However,in cases of indeterminate IHC results,molecular testing by MSI-PCR is preferred.Recently,NGS-based MSI assays are being widely used to detect MSI-H tumors.All three methods have high accuracy;however,the inconsistencies between them may lead to misdiagnosis.

Application of sintilimab combined with anlotinib hydrochloride in the clinical treatment of microsatellite stable colorectal cancer

BACKGROUND Microsatellite stable(MSS)colorectal cancer(CRC)is a common type of tumor with limited treatment options.Sintilimab and anlotinib hydrochloride are two extensively studied anticancer drugs.AIM To probe the clinical value of combining sintilimab with anlotinib hydrochloride in MSS CRC treatment.METHODS During the period spanning from April 2019 to April 2022,Zhejiang Provincial People’s Hospital accommodated a cohort of 92 patients diagnosed with MSS CRC who were classified into two distinct groups in our study,the observation group and the control group.The control group was administered anlotinib hy-drochloride as their designated therapy,whereas the observation group received the additional treatment of sintilimab in conjunction with the therapy assigned to the control group.The administration of treatment occurred in cycles consisting of a duration of 3 wk,and the evaluation of effectiveness took place subsequent to the completion of two consecutive cycles of treatment within both groups.A comparative analysis between the two groups was conducted to assess the short-term efficacy and ascertain the incidence of adverse events transpiring throughout the duration of the treatment period.Changes in the levels of carcinoembryonic Life Questionnaire-Core 30 were compared between the two groups prior to and subsequent to therapy.Finally,a 1-year follow-up was conducted for both groups of patients,and the survival status was recorded and analyzed.RESULTS The short-term effectiveness displayed by the observation group surpassed that exhibited by the control group,with a statistically significant discrepancy(76.09%vs 50.00%),reaching a significance level denoted as P<0.05.Following the administration of treatment,the observation group manifested a considerable reduction in numerous serum indicators,which were found to be lower than the corresponding pretreatment levels within the same group as well as the post-treatment levels observed in the control group(P<0.05).Post-treatment,the T lymphocyte subset levels within the observation group demonstrated a remarkable amelioration,surpassing the corresponding pre-treatment levels observed within the same group as well as the post-treatment levels observed in the control group(P<0.05).Subsequent to the therapeutic intervention,the observation group showcased a notable amelioration in the scores associated with multiple dimensions of life quality.These scores outperformed the pretreatment scores within the same group as well as the post-treatment scores observed in the control group(P<0.05).The safety levels of drug use in the two group were comparable(19.57%vs 13.04%),and no distinct difference was observed upon comparison(P>0.05).After the completion of treatment,both groups of patients underwent a 1-year follow-up outside the hospital.Throughout this period,1 patient within the observation group and 2 patients within the control group became untraceable and were lost to follow-up.During the follow-up period of the observation group,12 patients died,resulting in a survival rate of 73.33%(33/45),while in the control group,21 patients died,resulting in a survival rate of 52.27%(23/44).The implementation of Kaplan-Meier survival analysis revealed a conspicuous contrast in survival rates exhibited by the two groups(log-rank=4.710,P=0.030).CONCLUSION The combination of sintilimab and anlotinib hydrochloride demonstrated favorable efficacy in the treatment of MSS CRC patients,leading to improvements in patient immunity and prognosis.Additionally,it exerted inhibitory effects on the expression of carcinoembryonic antigen,CA199,and CA125.

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.

The Identification and Assessment on Genetic Characteristics in Grading Breeding Sheep Populations with Microsatellite Markers

[Objective] The aim of the research was to identify and assess the genetic characteristics of grading breeding sheep populations in Ba Yan Nur City. [Method] Genetic polymorphism and aggregation of seven sheep populations, including three breeding sheep populations (breeding F1, F2 and Bamei mutton sheep), three introduced mutton sheep breeds (Texel, Dorset and German Merino sheep) and one local female parent population (Mongolia sheep), were assessed using 10 microsatellite markers. [Result] By cluster analysis, the seven sheep populations can be divided into two groups. The F1 and German Merino sheep were closely related, which were clustered with F2, Bamei mutton sheep and Mongolia sheep to form one group while Texel and Dorset to form another group. The genetic aggregation of the seven breeds was assessed by Bayesian discrimination. And the results show that the genetic aggregation of F1 and F2 were lower while that of Bamei mutton sheep, Texel, Dorset and German Merino sheep were higher. [Conclusion] Better genetic stability has been formed in Bamei mutton sheep.

Microsatellite Variations Among Four Populations of Eriocheir sinensis

Using microsatellite markers, we analysed genetic polymorphism in four populations of Eriocheir sinensis, sampled from Jiangsu, Anhui, Liaoning and Tianjin. Twenty-four pairs of primers were used to amplify the target fragments ranging from 80 bp to 445 bp, which included 16 pairs designed in our laboratory and eight pairs published internationally. Two to 10 alleles per locus in four populations were amplified, and there were 155 alleles in all populations. The average number of alleles per locus was 6.458. The average number of effective alleles per locus （Ne） was 4.349l to 4.7234; the average observed heterozygosity （Ho） was 0.5690 to 0.6722; and the average expected heterozygosity （He） was 0.7238 to 0.7546. Hardy-Weinberg equilibrium analysis （Z2 test, P 〈 0.05） revealed that seven loci in the four populations were in equilibrium. The genetic distances between the four populations were calculated and revealed that the Anhui, Jiangsu and Tianjin crabs belong to a Yangtse River population, while the Liaohe crabs form another branch.

Comparative Microsatellite Analysis of Grass Carp Genomes of Two Gynogenetic Groups and the Xiangjiang River Group

The genomes of three groups of grass carp, namely the Xiangjiang River grass carp group （Xiangjiang group）, a one-generation artificially induced meio-gynogenetic grass carp group （meio-gynogenetic-1 group）, and a two-generation artificially induced meio-gynogenetic grass carp group （meio-gynogenetic-2 group）, were comparatively analyzed with microsatellite markers. Genetic polymorphism had been observed in the Xiangjiang group and most of the examined loci had more than two alleles. But the degree of genetic diversity was not very high. Although all the examined genetic loci in the analyzed individuals were in homozygous state, the genotypes of different individuals of the group were not identical in the meio-gynogenetic-1 group. In the meio-gynogenetic-2 group, not only the examined genetic loci of each individual were homozygous but also the genotypes of all the analyzed individuals of the group were the same. These results suggested that the examined meio-gynogenetic-2 group is a homozygous group and homozygous clone could be produced by continuous artificial induction of gynogenesis for two generations. It was found that the polymorphism existed not only at the allele level but also at the locus level; many alleles of the microsatellite loci and some of the microsatellite loci had been lost during the process of artificial gynogenesis. Therefore, both protection of the diversity of natural grass carp resource and selection of homozygous traits with desired economic genotypes are very important aspects for grass carp breeding.

Genetic Quality Analysis of Xiphophorus helleri RR-B Strain Using 18 Microsatellite Loci

[Objective] The study aimed to further understand the genetic quality of Xiphophorus helleri RR-B strain and estimate the feasibility of microsatellite markers in genetic monitoring of aquatic laboratory animals.[Method] Eighteen microsatellite loci which had polymorphism in wild X. helleri were used for genetic quality analysis of RR-B strain (red eyes and red body) by PCR. [Result] All these 18 microsatellite loci displayed single allelic gene band in 30 individuals of X. helleri RR-B strain and the average homozygote rate reached 100%. [Conclusion] These loci could be used as markers in genetic monitoring of X. helleri RR-B strain, which had obtained fairly high genetic homozygosity by inbreeding for more than 20 generations. This study provided basic data for establishing the molecular genetic monitoring standard of aquatic laboratory animals.

Microsatellite DNA Marker Analysis of Genetic Diversity in Wild Common Carp (Cyprinus carpio L.) Populations

Thirty microsatellite loci were used for analyzing six wild populations of common carp （Cyprinus carpio L.）. Observed （Ho） and expected （He） heterozygosity values, polymorphic information content （PIC）, and number of effective alleles （Ae） were all detected. Genetic similarity index and genetic distance were computed based on the allele frequency. The Hardy-Weinberg Equilibrium was checked according to the test of χ^2. Genetic differentiation and hierarchical partition of genetic diversity were evaluated by FST and Nm. A clustering dendrograrn was made based on the results of UPGMA methods using the PHYLIP software package （version 3.63）. There were totally 8,136 fragments ranging from 125 bp to 414 bp in length. Three to thirteen alleles were amplified in 30 loci and 210 alleles in all six populations. The average number of alleles in each locus was seven. The result showed that 1） the level of genetic variability was moderate in the six populations. Polymorphic information contents of the six wild common carp populations were 0.44, 0.52, 0.53, 0.57, 0.63, and 0.64 respectively. Effective alleles were from 1.04 to 4.72, the average numbers in each population were 2.19, 2.60, 2.42, 2.43, 2.45, and 2.33. The average expected heterozygosity values were 0.50, 0.59, 0.56, 0.56, 0.57, and 0.54 respectively; 2） the highest genetic similarity index that came from the populations of BR and ZL was 0.8511 and the lowest index was 0.6688, and it came from the populations of BR and HN. There was a correlation between the clustering result and the geographical distribution.

Genetic Diversity of Microsatellite DNA Loci of Tibetan Antelope(Chiru,Pantholops hodgsonii)in Hoh Xil National Nature Reserve,Qinghai,China

The Tibetan antelope （Pantholops hodgsonii）, indigenous to China, became an endangered species because of considerable reduction both in number and distribution during the 20th century. Presently, it is listed as an Appendix Ⅰ species by CITES and as Category I by the Key Protected Wildlife List of China. Understanding the genetic diversity and population structure of the Tibetan antelope is significant for the development of effective conservation plans that will ensure the recovery and future persistence of this species. Twenty-five microsatellites were selected to obtain loci with sufficient levels of polymorphism that can provide information for the analysis of population structure. Among the 25 loci that were examined, nine of them showed high levels of genetic diversity. The nine variable loci （MCM38, MNS64, IOBT395, MCMAL TGLA68, BM1329, BMSI341, BM3501, and MB066） were used to examine the genetic diversity of the Tibetan antelope （n = 75） in Hoh Xil National Nature Reserve（HXNNR）, Qinghai, China. The results obtained by estimating the number of population suggested that all the 75 Tibetan antelope samples were from the same population. The mean number of alleles per locus was 9.4 ± 0.5300 （range, 7-12） and the mean effective number of alleles was 6.519± 0.5271 （range, 4.676-9.169）. The observed mean and expected heterozygosity were 0.844 ± 0.0133 （range, 0.791-0.897） and 0.838 ± 0.0132 （range, 0.786-0.891）, respectively. Mean Polymorphism Information Content （PIC） was 0.818 ± 0.0158 （range, 0.753-0.881）. The value of Fixation index （Fis） ranged from -0.269 to -0.097 with the mean of -0.163 ± 0.0197. Mean Shannon＇s information index was 1.990 ± 0.0719 among nine loci （range, 1.660-2.315）. These results provide baseline data for the evaluation of the level of genetic variation in Tibetan antelope, which will be important for the development of conservation strategies in future.

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.

Isolation of CA/GT Microsatellites from the Paralichthys olivaceus Genome

A library rich in CA/GT microsatellites was constructed from the Paralichthys olivaceus genome by combining biotin capture method and radioactive labeling hybridization. Five hundred and twenty six positive clones were obtained through twice screens. Sequencing confirmed 133 microsatellite loci （number of repeats t〉 5） in 119 positive clones. Of these microsatellites, two （1.5%） had compound repeat motifs, 63 （47.37%） had perfect motifs and 68 （51.13%） had imperfect motifs. Primer pairs were designed in the flanking regions of 22 microsatelites and subjected to PCR amplification. In 8 artificial gynogenesis families, four pairs failed to amplification, one pair was monomorphic, and the rest were polymorphic with an average of 5.2 alleles per locus. Heterozygosities ranged between 0. 375 and 0. 846, PIC ranged between 0. 305 and 0. 823. The results suggested that most of the microsatellites we isolated were qualified to be applied to the population genetic studies of P. olivaceus.

Searching for Protein-coding Genes Using Microsatellites in Common Carp by Comparing to Zebrafish EST Database

In this study, an in silico approach was utilized to identify homologies existing between common carp microsatellite sequences and GenBank database using Blastn and Blastx searches. About 875 microsatellite sites with flanking sequences over 50bp of common carp were first compared to the zebrafish EST database. The results showed that 121 homologies were found using Blastn. Subsequent Blastx searches confirmed 94 sites recorded in the protein database. Except for 33 hypothetical proteins and three unknown proteins, seven out of 58 characterized proteins have been mapped to two linkage maps. In addition, two polymorphic STS markers were developed using matched zebrafish EST sequences by PCR-SSCP method, of which one marker HLJZe33 was mapped successfully. This study was a pilot for comparative studies between common carp and zebrafish, and the results demonstrated that more genetic and genomic resources of zebrafish can be used for the genome research of common carp.