Genetic Polymorphism of Ten Microsatellites in Two Goat Breeds and Its Relationship with Heterosis

[Objective] This study aimed to investigate the genetic diversity of Macheng black goat and its correlation with heterosis.[Method] Ten microsatellite markers were selected for polymorphism investigation and statistical analysis of Boer goat and Macheng black goat populations.[Result] The results showed that totally 175 alleles were found in 10 microsatellite loci; to be specific,the maximum number of detected alleles was 23,and the minimum number was 10; the effective number of alleles （Ne） was 6.4-18.1,with absolute difference value of 1.6-8.1 from the observed number of alleles.The highest gene frequency was 0.239 1 and the lowest was 0.002 7.The polymorphic information contents of all the ten microsatellite markers were above 0.95.The observed heterozygosity （Ho） ranged from 0.616 7 to 0.984 4 and the expected heterozygosity （He） ranged from 0.844 1 to 0.944 6.The average expected heterozygosity of Boer goat and Macheng black goat was respectively 0.894 0 and 0.906 7.Various body weight and body size indices of Boer goatxMacheng black goat hybrids were improved in varying degrees compared with Macheng black goat （with an increase range of 0.32％-30.06％）.The average heterosis rates of body height and chest girth were relatively high,while average heterosis rate of body weight was relatively low.[Conclusion] The genetic distance between Boer goat and Macheng black goat was 0.379 5,which is consistent with the geographical distribution of Boer goat and Macheng black goat populations and is fully relevant to the heterosis of Boer goat × Macheng black goat hybrids,indicating that investigating polymorphism via microsatellite loci is one of the feasible means to predict and analyze heterosis between varieties.

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 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.

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.

Polymorphism analysis of microsatellites and construction of linkage map in part regions of four chromosomes in chicken

Based on chicken＇ consensus map issued in 2000, 17 microsatellites near 4 candidate genes such as IGF2, OBR, GDF8 and APOA 1 in 4 chromosomes （chromosome 5, 7, 8 and 24） were chosen for polymorphism analysis and construction of linkage map. Combining the technique of PCR and the fluorescent semi-automated detection, genome scanning was performed for 440 chickens, which was derived from China Agricultural University chicken resource families within three generations. The individuals of this resource families were genotyped. The results showed that the number of alleles ranged from 4 to 14; heterozygosity （H） of markers was between 0.3116 and 0.9148. Polymorphic information content （PIC） varied from 0.2672 to 0.8679. Microsatellites along with above-mentioned 4 candidate genes doing as general markers were used to construct linkage map. The spans of 4 linkage maps constructed in the part region of chromosome 5, 7, 8 and 24 were 263.5, 79.9, 206.2 and 104.2 cM, respectively. The order of markers was consistent with that of counterpart of reported consensus map. However, The spans of linkage map were larger than that of consensus map. The constructed linkage maps laid the foundation for mapping quantitative trait loci （QTL） responsible for economically important traits in chicken.

Correlation study between the polymorphism of repetitive sequence in gene CAG of androgen receptor and the occurrence and progression of prostate cancer

Objective:To explore the relation between the polymorphism of repetitive sequence in gene CAG of androgen receptor(AR)and the susceptibility and clinical stages as well as pathological grading of prostate cancer among Han population.Method:Sixty-eight cases with prostate cancer hospitalized in Urinary Surgery Department from Feb.2010 to Feb.2012 and 60 healthy cases were chosen as research subjects.Methods of PCR and direct sequencing were adopted to detect DNA sequence of AR gene and the length of repetitive sequence in CAG.Results:The lengths of repetitive sequence in CAG of patients with prostate cancer and healthy people were(22.3±4.6)and(23.0±4.9),respectively showing no statistical significance.Comparing length(repetitive sequence of CAG)>22,those with that<22 suffer a remarkably higher risk of prostate cancer(P<0.05).The number of repetitive sequence in CAG of patients at clinical stage C-D was less than that of patients at stage B,and the number of repetitive sequence in CAG of patients with poorly differentiated prostate cancer was also less than that of patients with moderately and highly differentiated prostate cancer.But there was no statistical significance int the difference(P>0.05);the proportion of patients with length<22 at clinical stage C-D was much larger than that of patients at clinical stage B(P<0.05),and as the aggravation of pathological grading,the proportion of patients with the length<22 was also remarkably increased and there was significant difference between patients with highly differentiated prostate cancer and those with poorly differentiated prostate cancer(P<0.05).Conclusions:There is correlation between the occurrence and development of prostate cancer in Han population and the polymorphism of repetitive sequence in gene CAG of androgen receptor.The less the number of repetitive sequence in CAG is,the higher the risk of prostate cancer will be and the more severe the clinical stage and pathological grading will be.

Development of 57 Novel Polymorphic Microsatellite Markers in Half-Smooth Tongue Sole(Cynoglossus semilaevis)

Half-smooth tongue sole (Cynoglossus semilaevis) is a promising species for aquaculture in China.The wild population of C. semilaevis is under threat from environmental factors. Microsatellite markers are very suitable for assessing genetic diversity. Four microsatellite-enriched libraries of half smooth tongue sole (Cynoglossus semilaevis) were constructed,from which 57 polymorphic microsatellites were isolated and characterized.The polymorphism of these microsatellites was assessed by genotyping in 30 individual fish.The number of alleles ranged from 2 to 11, with an average of 4.614 alleles per locus.The values of observed and expected heterozygosities ranged from 0.1000 to 1.0000 and from 0.0966 to 0.8847 respectively. Polymorphism information content (PIC) ranged from 0.0905 to 0.862.These markers would be useful for population structure assessment,genetic linkage map construction and parentage analysis for this species.

Study on relationship between polymorphism of hAR gene (CAG)n micro-satellite and prostate cancer

Objective: To study the relationship between the polymorphic (CAG)n micro-satellite of human androgen receptor (hAR) gene and prostate cancer (PCa). Methods: The number of (CAG)n repeats in 107 normal males were measured by a two-step [α-32P]-dCTP incorporated asymmetric polymeric chain reaction (PCR), and the (CAG)n repeats of both malignant and nonmalignant prostate cells in fixed paraffin-embedded tissue (PET) specimen from 36 case of PCa were determined by sequence analysis. Results: The repeats of polymorphic (CAG) n among normal men ranged from 11 to 29, and the most frequent repeat was 22(18. 69%), with 23(14. 02%), 24(10. 28%) and 21(10. 28%) being less frequent. The (CAG)n repeats of malignant prostate cells equaled to that of nonmalignant adjacent prostate tissue cells from the same PET specimen in all 36 PCa, and the (CAG)n repeats in 36 PCa which ranged from 16 to 22 were shorter than that in normal males significantly (P<0. 05), while no significant difference in (CAG)n repeats among various grade of tumor's differentiation (well-differentiated, intermediate-differentiated and poor-differentiated) was found (P>0. 05). Conclusion; The present study suggest that short hAR gene (CAG)n micro-satellite might be associated with the occurrence of PCa, but not with the differentiation of PCa.

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.

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.

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 and Characterization of Microsatellite DNA Markers from Forest Musk Deer(Moschus berezovskii)

In this report, we describe the isolation and characterization of microsatellite loci for forest musk deer （Moschus berezovskii） through an improved enrichment protocol. Five new markers were isolated from the genomic DNA of forest musk deer and showed high polymorphism with 4 - 13 alleles in 24 sampled individuals from the population of Jinfeng Mountain, Sichuan Province, China. The observed and expected heterozygesities were from 0.429 - 0.957 and 0.587 - 0.902, respectively. The average polymorphism information content （PIC） value in these five loci was 0. 730. This suggests that the five microsatellite loci are a valuable tool for further studies about forest musk deer.

Isolation of Microsatellite DNA and Preliminary Genomic Analysis of Mud Carp (Cirrhina molitorella)

In order to determine the applicability of microsatellite primers developed from common carp （ Cyprinuscario ） for genomic analysis in mud carp （ Cirrhina molitorella ）, 24 primer pairs from common carp were designed to amplify microsatellite loci in the mud carp containing CA, GA, AT and GGGA sequences. Thirteen primers （54%） successfully amplified specific products in the mud carp and 11 primers （48%） showed high polymorphism in the mud carp population. The results indicated that the average number of alleles per locus in the mud carp stocks was 5.2. Average heterozygosity （Ho）, unbiased expected heterozygosity （He） and polymorphism information content （PIC） in the wild population were 0.61 ± 0.2, 0.8 ± 0.09 and 0.72 ± 0.1 respectively. Several Hardy-Weinberg departure value were significandy departed from Hardy-Weinberg equilibrium. The study showed that microsatellite primers from a species of Cyprinidae can be used for mud carp genetic analysis without much cost or time input.

Cross-species amplification and characterization of microsatellite DNA loci from Gallus gallus in Bambusicola thoracica

The Chinese Bamboo Partridge(Bambusicola thoracica) is a gamebird endemic to China.Ten polymorphic microsatellite(simple sequence repeat) markers were obtained through cross-species amplification for this partridge from its relative species Gallus gallus.The number of alleles per locus varied from 4 to 13.The observed heterozygosity ranged from 0.1220 to 1.0000 and the expected heterozygosity from 0.1183 to 0.8898.Four microsatellite loci showed significant differences from the Hardy-Weinberg equilibrium.These polymorphic loci provide a valuable tool for the investigation of the phylogeography and conservation genetics of this partridge.

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.

A New Problem with Cross-Species Amplification of Microsatellites: Generation of Non-Homologous Products

Microsatellites have been widely used in studies on population genetics, ecology and evolutionary biology. However, microsatellites are not always available for the species to be studied and their isolation could be time-consuming. In order to save time and effort researchers often rely on cross-species amplification. We revealed a new problem of microsatellite cross-species amplification in addition to size homoplasy by analyzing the sequences of electromorphs from seven catfish species belonging to three different families （Clariidae, Heteropneustidae and Pimelodidae）. A total of 50 different electromorphs were amplified from the seven catfish species by using primers for 4 microsatellite loci isolated from the species Clarias batrachus. Two hundred and forty PCR-products representing all 50 electromorphs were sequenced and analyzed. Primers for two loci amplified specific products from orthologous loci in all species tested, whereas primers for the other two loci produced specific and polymorphic bands from some non-orthologous loci, even in closely related non-source species. Size homoplasy within the source species was not obvious, whereas extensive size homoplasy across species were detected at three loci, but not at the fourth one. These data suggest that amplification of products from non-orthologous loci and appearance of size homoplasy by cross-amplification are locus dependent, and do not reflect phylogenetic relationship. Amplification of non-orthologous loci and appearance of size homoplasy will lead to obvious complications in phylogenetie interference, population genetic and evolutionary studies. Therefore, we propose that sequence analysis of cross-amplification products should be conducted prior to application of cross-species amplification of microsatellites.

Role of detection of microsatellite instability in Chinese with hereditary nonpolyposis colorectal cancer or ordinary hereditary colorectal cancer

AIM： To detect microsatellite instability （MSI） in patients with hereditary nonpolyposis colorectal cancer or ordinary hereditary colorectal cancer and to provide criteria for screening the kindreds with hereditary nonpolyposis colorectal cancer at molecular level.METHODS： MSI was detected in the specimens from 20 cases with HNPCC, 20 cases with ordinary hereditary colorectal cancer and 20 cases with sporadic colorectal cancer by means of polymerase chain reaction-single strand conformation polymorphism. RESULTS： The positive rate of MSI was 85% （17/20） in HNPCC group, 40% （8/20） in ordinary hereditary colorectal cancer group and 10% （2/20） in the sporadic colorectal cancer group respectively. The differences were significant. The mean ages of the three groups were 43.6, 52.2, and 61.8 years respectively, which increased gradually. The incidence of right hemicolon cancer was 64.7%, 37.5%, and 0% respectively, which decreased gradually and had significant difference. The expression ratio of BAT26 and BAT25 was 94.1% respectively, which was highest in the 5 gene sites studied. The incidence of poorly differentiated adenocarcinoma was 70.6% in HNPCC group among high frequency microsatellite instability （MSI-H）, which was higher than the other two groups, which had 50% and 50% respectively. CONCLUSION： The incidence of MSI-H is higher in HNPCC group. The detection of MSI is simple and economical and has high correlation with the clinicopathologic feature of HNPCC and can be used as a screening method to detect the germ line mutation of the mismatch repair gene.

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

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

Profile and development of microsatellite primers for Acanthogobius ommaturus based on high-throughput sequencing technology

Acanthogobius ommaturus,a fish species of the Family Gobiidae,is a marine commercial fish perched on the bottom of seawater.In this study,Illumina high-throughput sequencing technology was applied to obtain the candidate microsatellite markers of A.ommaturus.A total of 4746 microsatellite-rich fragments were found,of which 4542 microsatellites are with primer fragments,containing 971 dinucleotide sequences,2643 trinucleotide sequences,569 tetranucleotide sequences,406 pentanucleotide sequences,and 212 hexanucleotide sequences.Based on the results of high-throughput sequencing,a total of 141 pairs of the microsatellite primers were designed and screened.And then 24 polymorphic primers were finally obtained by polyacrylamide gel electrophoresis.In total,271 alleles were detected in the 24 pairs of primers.The number of alleles for different primers ranged from 5 to 19.The average number of effective alleles(Na)was 11.292;the average observed heterozygosity(Ho)of the 24 pairs of primers was 0.665,the average expected heterozygosity(He)was 0.880,and the average polymorphic information content was 0.846.All sites were highly polymorphic(PIC>0.50).

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

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