| Genomic microsatellites(simple sequence repeats;SSRs),iterations of 1-6 bp nucleotide motifs,have been detected in the genomes of every organism analysed so far.The Rich polymorphism of microsatellites was due to its high degree of variation which causing amount of alleles.The differences between alleles were only the number of repeat units and the flanking sequences of microsatellite DNA were conservative.Therefore,when a microsatellite locus was isolated and characterized,we could design a pair of oligonucleotides primers basing on its flanking sequence and then identified the composition and polymorphism of this locus by differentiating the length of the different allele fragments represented through the agarose electrophoresis after PCR amplification.Alleles of microsatellite DNA is dominant inheritance and in accordance with Mendel's law,in addition,microsatellite DNA can steadily pass on between parental and offspring and natural selection show a smaller effect on its genetic diversity.All these advantages made microsatellite DNA were widespread applied in species' genetic diversity research,genetic linkage map construction,phylogeny and paternity assignment.Currently,the most common methods for obtaining microsatellite include:searching the existing microsatellite loci from Public DataBase;cross-species microsatellite amplification and enrichment the genomic library for microsatellite loci.However,a pre-sequencing SSR enrichment step is a necessity for obtaining microsatellite DNA in avian which SSRs are rare in the genomes of some species.In this study,we constructed a microsatellite enriched genomic library for Parus vairus with an appropriate probe containing inserts with(AC)15 repeats.Loci which displayed high levels of polymorphism were applied in extra-pair paternity assignment together with loci that obtained from cross-species amplification.Thus would supplement the genetic information of Parus vairus and complete the mechanism of its extra-pair copulation and dispersal.Four genomes of Parus vairus were used to screening the microsatellite loci.Approximately 68 positive clones were screened and 11 clones had sufficient flanking sequences for designing primers.Results of PCR amplification revealed that 7 primer pairs could consistent specific products.48 Parus vairus samples were evaluated for the 7 primer pairs and results showed that 4 primer pairs were polymorphic and well amplificated at each sample.The polymorphic information content(PIC)ranged from 0.728 to 0.823 and the mean polymorphic information content was 0.793.The numtber of alleles(K)ranged from 11 to 17,the average number of allele was 13.5;the values of expected heterozyosity(He)ranged from 0.751to 0.851,the average expected heterozygosity(He)was 0.819.Three out of four microsatellite loci and seven loci obtained from cross-species amplification were selected to assign the paternity for Parus vairus in 2013.We established paternity in all 98 individuals of 12 broods by microsatellite genotyping.The genotype data revealed a reasonable amount of polymorphism.The average expected heterozygosity(He)was 0.816 and the mean PIC in our samples was 0.796.The combined exclusion power for the 10 microsatellite loci was 99.97%for Excl 1 and 99.99%for Excl 2 which demonstrated that these 10 microsatellite loci could accurately assign paternity for Parus vairus.Paternity results show that 5 of the 12 broods(41.67%)occured extra-pair offspring and accounted for about 14.86%(11/74)of all offspring.Although the level of extra-pair paternity was similar with the results that simply got from loci which obtained by cross-species amplification,we also provide new evidence for other genetic studies on Parus varius such as comparison of genetic diversity among subpopulation. |
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