Preliminary Study on Applicability of Microsatellite PrimersDeveloped from Common Carp for Genomic Analysis of Grass Carp
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Abstract:
In order to determine the applicability of microsatellite primers developed from common carp (Cyprinus carpio) for genomic analysis in grass carp (Ctenopharyngodon idellus), twentyeight pairs of common carp primers designed for microsatellites containing CA motifs were employed to amplify the microsatellite loci in the genome of grass carp. The conditions of polymerase chain reaction (PCR) were optimized for the fidelity of DNA synthesis during PCR amplification. Two kinds of nonspecific PCR products, heteroduplex bands and shadow bands, were eliminated successfully by decreasing the extension temperature and Mg2+ concentration. 7 primers (about 25%) amplified specific products successfully and 4 primers (about 14.3%) have shown the polymorphism in a small population of the wild grass carp of xiangiang river (only 8 samples of fish). This result indicates that there are about 50% homology of sequences flanking the microsatellite loci between the common carp and grass carp, and some of the common carp microsatellite primers can be used for grass carp genetic analysis without much costing and time consuming.Keywords:
Grass carp
Common carp
genomic DNA
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Objective: This study used the microsatellite primers of carp to isolate Carassius auratus L's new microsatellite markers,then analyzed the polymorphism of the two microsatellite loci.Methods: 6 pairs of microsatellite primers were used to amplify the genomic DNA in Carassius auratus L using the PCR technique,The PCR products were analyzed by 8% non-denatured polyacrylamide gelatin electrophoresis,and stained with silver,then we identified the sequences of 2 pairs of primers's PCR products which contain microsatellite DNA fragments.Results:After the sequences aligning and BLAST analysis,the results indicated that we isolated 2 new microsatellite sequences of Carassius auratus L,which contains the repeat units of TA and AC.Then we designed two primers according to the new microsatellite sequences and further amplified by PCR on C.auratus auratus to investigate polymorphism of the 2 microsatellite markers.The results show that the heterozygosis of the two microsatellite loci are 0.611 and 0.644,the polymorphism content are 0.536 and 0.572.Conclusion:which indicated that these two microsatellite loci have high polymorphism.The results indicated that the microsatellite markers of Carassius auratus of great importance in heredity multiplicity,heredity chain-like atlas construction,the molecular mark assistance breeding and the identify of idioplasm resources.
Carassius auratus
genomic DNA
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Microsatellites are important markers for development of genetic maps,germ plasma assessment,quantitative trait loci mapping because of their high polymorphism,abundance,co-dominance,and small locus size.In aquatic species,microsatellite markers have been used for salmon,tilapia,rainbow trout,catfish etc.on genetic map,quantitative trait loci mapping,and population genetics assessment.But only a few local aquaculture species in China,their microsatellite markers have been developed.Because up till now there are not enough micrksatellite markers,the technique of RAPD marker is still used in genetic assessment for some important aquatic species such as river crab,common carp,silver crucian carp,silver carp etc.For genetic assessments research,the marker that identifies different populations in one species is very important.Owing to its co-dominant property,microsatellite marker should be a good candidate for this kind of marker.In this test,some microsatellite sequences for common carp were cloned by two methods.One was that traditional inserted DNA fragments libraries were screened by plaque hybridization using oligos (CA)_15 as probes,end-labeled with [γ-32] ATP.The other was that inserted DNA fragments were linked onto linkers and then enriched microsatellite sequences with magnetic beads which was linked with biotinylated (CA)_15 probes,finally the PCR-based library was made.2000 colones from the former method were selected and screened,and 45 colones were positive.In those positive colones,22 microsatellite sequences were gotten.Among them,perfect was 63.6%,imperfect was 22.7%,compound was 13.7%,repeated numbers over 10 were 9 and about 40.9%.About 2600 colones from the latter method were selected and screened,and 1300 colones were positive.From those positive colones,390 colones were sequenced,and 314 microsatellite sequences were gotten.In those microsatellites,perfect was 79.0%,imperfect was 14.3%,compound was 6.7%,and repeated numbers over 10 were 293 and about 93.3%.This result showed that the magnetic beads enriched method could be a high efficiency and low cost method,and microsatellite sequences from it could be of high quality.So,we recommended that magnetic beads enriched method be used in making microsatellite markers.
Molecular marker
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The genomic DNA from common grass carp (PC) and allogynogenesis grass carp F1 (CC) induced by inactivate sperms of squaliobarbus curriculus were isolated. Using RAPD and SCAR technology, 16 primers with high repeatability and stability were selected from the 100 RAPD random primers, and 7 specific DNA fragments had been discovered. One of the fragments was specific in allogynogenesis grass carp F1, while the others only appeared in common grass carp variety. After purification, cloning and sequencing, only two specific fragments amplified by S32 and S336 had been obtained. Based on their sequence information, SCAR primers were designed. However, the PCR products amplified by S336-Scar1F/S336-Scar1R were the same between two populations, and so were SCAR primers S336-Scar2F/S336-Scar2R. Fortunately, there was difference between common grass carp variety and allogynogenesis grass carp F1 varity when amplified by S32-Scar1F/S32-Scar1R and S32-Scar2F/S32-Scar2R. 115 individuals originated from 2 populations, including 55 CC and 60 PC, were used to verify the reliability of the SCAR maker S321643 (amplified by S32-Scar1F/S32-Scar1R). The results showed S321643 appeared in 46 PC individuals (76.7%), while disappeared in all 55 CC individuals (0%).
Grass carp
Common carp
genomic DNA
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Microsatellite-enhanced libraries of AC-repeats and GATA-repeats in Procypris rabaudi were constructed using repeat-enrichment method with biotin-labeled oligos and streptavidin magnetic beads.The positive clones were screened through PCR method using oligoA and(AC)12or oligoA and(GATA)6 as primers.The ratios of positive clones of(AC)n and(GATA)n microsatellite-enhanced libraries were about 30% and 7%,respectively.Total 61 microsatellite sequences were obtained by sequencing 40 AC-repeats positive clones and 30 GATA-repeats positive clones,which included one trinucleotide repeat microsatellite sequence(TGA).19 pairs of primers for AC-repeats microsatellites,16 pairs of primers for GATA-repeats microsatellites and one pair of primers for TGA-repeats microsatellites were designed.Legible and constant expected DNA products could be amplified using 20 pairs of the primers through PCR optimization.In order to check these microsatellite loci whether can be used for relative species,then the 20 pairs of primers were used to amplify microsatellite sequences of Spinibarbus sinensis,and legible and constant expected products could be obtained using 12 pairs of the primers.Therefore,the 20 microsatellite loci of Procypris rabaudi obtained from the present study are potential for further investigations of genetic diversity and population structure in Procypris rabaudi and relative species.
Direct repeat
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Microsatellite DNA was isolated from goldfish with combining biotin capture method. We obtained 38 positive clones and isolated 31 microsatellite sequences. 12 pairs of primers were designed by Primer Premier 5. 0 and 8 pairs of primers can obtain amplifying products among them. The genetic diversity of the artificial breed goldfish populations were studied by using the 8 Microsatellite primers. The results showed that the genetic diversity level of two populations were high. The average heterozygosity( Ho) in two populations was from 0. 534 3 to 0. 565 5, average excepted heterozygosity( He) was from 0. 628 6 to 0. 657 0,and average polymorphism information content( PIC) was from 0. 500 9 to 0. 510 6. The genetic distance was 0. 095 7 and genetic identity was 0. 921 1. All of 8 microsatellite locus could be used for genetic analysis in goldfish.
Primer (cosmetics)
Genetic distance
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Summary Forty‐eight primer‐pairs complementary to unique DNA sequences flanking chicken (genus Gallus ) genomic (TG) n microsatellite repeats were previously designed. These primer‐pairs were used in the polymerase chain reaction to amplify turkey (genus Meleagris ) genomic DNA loci. Results indicated that the majority (92%) of these primer‐pairs generated amplification products in turkey genomic DNA. Hybridization using end‐labelled (TG) 8 as a probe showed that, out of 41 primer‐pairs tested, only 14 generated an amplification product that also contained a detectable (TG) n microsatellite repeat when turkey DNA was the template. Among 18 primerpairs tested for polymorphism, using three commercial turkey lines, five were found to exhibit length polymorphism, three of which did not contain a detectable TG repeat. Therefore, a significant portion of chicken microsatellite markers can be useful for genomic mapping and linkage analysis in the turkey, reducing the costs involved in producing turkey‐specific microsatellite markers.
Primer (cosmetics)
genomic DNA
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AIM:To construct microsatellite-containing library of Anopheles sinensis and to isolate and characterize the polymorphic microsatellite loci.METHODS:Genomic DNA fragments were hybridized with biotinylated oligonucleotide probes.The hybridized fragments were captured with Vectrex Avidin D and enriched by centrifugalization with ultra-4-column ultrafiltrate.The target fragments were amplified,cloned and sequenced.The suitable microsatellite loci were chosen in An.sinensis's library to establish PCR amplification assay.The polymorphism screening was conducted by PAGE gel electrophoresis with An.sinensis field populations.RESULTS:An enrichment protocol yielded 252 microsatellite sequences,their GenBank Accession Numbers were from EF620047 to EF620298.There were the most percentage of dinucleotide repeat,the more of tri-nucleotide repeat and fewer of multi-nucleotide repeat.The(CA)n and(GT)n were higher abundance in the microsatellite DNA libary.The number of motif repeate was from 2 to 54.The percentage of perfect microsatellite DNA sequence was 35.3%,imperfect sequence was 20.2% and compound sequence was 18.7%,and the rest was non-typical microsatellite sequence.We designed primers to amplify 22 unique microsatellites,and 20 of which amplified successfully.A survey of 30 individuals showed that 15 loci were highly polymorphic.CONCLUSION:A total of 15 polymorphic microsatellite loci of An.sinensis are first reported.These markers will be useful for population genetic studies and genome mapping in An.sinensis.
genomic DNA
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Objective:The primers originated from common carp are applied into population genetics of grass carp.But no polymorphism in length is detected.Some methods are applied to improve the useness of these primers.Methods:The arbitrary primers of TRAP and microsatellite primers are combined for amplification of the flank sequence of microsatellite sequences of Ctenopharyngodon idella and SSCP has also been applied for further analysis of these microsatellite sequences,Result:Relatively high polymorphism in length is detected by combination of arbitary primers from TRAP and microsatellite primers.Some changes are also found in nucleic acid sequences and different gene types can also been distinguished by SSCP.Condusion:TRAP and SSCP is a useful method in research work of population genetics.Some microsatellite sequences without polymorphism in length are also used in population genetics.
Single-strand conformation polymorphism
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RAPD-PCR was performed with 200 random primers on DNA samples of Heilongjiang wild carp(Cyprinus carpio haematopterus Temminck et Schlegel),frigid-resistance strain of Red purse carp(Cyprinus carpio var.wananensis),Red purse carp(Cyprinus carpio var.wananensis),Boshi carp(Cyprinus pellegnini Tchang),cross F_(2) of frigid-resistance strain of Red purse and Boshi carp,respectively.RAG20 was identified to be a molecular marker which associated with cold tolerance traits of common carp(cyprinus carpio).Examination to F_(2) segregated individuals verified that this molecular marker was reliable.Up to now,10 molecular markers associated with cold tolerance of common carp were obtained,and the marker 5N1451c interrelated with cold tolerance Cyprinus (Cyprinus) pellegrini Tchang has been mapped on linkage 5.It further demonstrates that cold tolerance trait is a quantitative trait loci(QTL),which is controlled by small multi-genes.In addition,specific fragments from four primers above were extracted from the agarose and purified respectively.The purified products were then ligated to pMD 18-T vector which were transformed to competent E.coli DH 5α for sequencing.The sequencing of three specific fragmens has been finished.The length of the amplified fragments was 1?021?bp,422?bp,1?043?bp respectively.The parpose of this study is to redesign primers upon the sequences and convert RAPD markers into the stable SCAR markers finally.
Common carp
Molecular marker
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Total DNA was extracted from tail muscles of Fenneropenaeus chinensis. DNA quality controls were per-formed using agarose gel electrophoresis and only fragments at 500 - 1000 bp were used for constructing a small-size fractionated genomic libraries. A genomic DNA library using the bacterial artificial chromosome (BAC) have been constructed in which the Genomic DNA digested by Sau3A I was ligated to the BAC vector and the ligation mixture was used to transform the bacterial strain DH5α.By using the on-line bioinformatic software Repeatmasker, small-size fractionated genomic libraries of F. chinensis were screened. Twenty-eight microsatellite sequences were ob-tained,according to which 28 pairs of primers were designed using computer program Primer Premier ( Version 5.00). The optimal results were obtained by optimizing quantity of reagents and reaction conditions. There were 14 primers whose PCR products can be detected by electrophoresis,among which five microsatellite loci were polymor-phic in mixed templates (eight Chinese shrimp samples) . In these five microsatellite loci,genetic diversity was anal-ysed by electrophoresis analysis in a cultured population consisting of 20 Chinese shrimp F. chinensis samples. The allele numbers, the expected heterozygosity and the PIC values of them in the locus RS0871 were low. Except for the locus RS0871 the alleles numbers distributed from 6 to 8, and the range of these alleles were between 142 bp and 364 bp,similar to their predicted size. The expected heterozygosity and the PIC values of them distributed from 0.757 7 to 0.806 4 and 0.7180 to 0.770 9,respectively. These values showed that 4 microsatellite loci except for the locus RS0871 could be applied for genetic analysis. This result indicated that polymorphic chang of microsatellite primers was related to the core repeats motifs of the microsatellites. As a result, there will be less costing and time consuming in primer designs according to the core repeats motifs of microsatellite. Moreover, the microsatellite markers will be useful for further studing on accessions identification and breeding of F. chinensis, which would provide some valu-able tools for marker-assisted selection breeding and gene mapping in F. chinensis.
genomic DNA
Agarose gel electrophoresis
Primer (cosmetics)
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