Development and Application of EST-SSR Marker in Asplenium nidus

doi:10.7668/hbnxb.2015.06.020

Abstract

Abstract: To accelerate the application of molecular markers in Asplenium nidus ,EST-SSR markers development and utilization form the Asplenium nidus EST sequences were performed.A total of 42 907 EST sequences were obtained from the Asplenium nidus transcriptome,and these sequences were screened by using MISA software to search for SSR motifs.Forty pairs of primers were designed by Primer 3.0 software,and the polymorphism of the primer was tested by PCR amplification of 10 ferns.A total of 6 067 EST-SSR were identified with an average of one SSR per 6.62 kb,and included 106 SSR motif types.Dinucleotide repeats was the dominant type(2 873,47.35%),followed by trinucleotide(1 107,18.25%)and mononucleotide(972,16.02%)repeats.The dominant repeat motif in all EST-SSRs was AG/TC(1 371,22.60%),followed by CA/GT(1 066,17.57%)and A/T(628,10.35%).40 pairs of EST-SSR primers were designed and synthesized based on different motifs types,and verified with 10 fern varieties for polymorphism.Among them,26 primer pairs could produce clear and stable bands,and 17 out of 26 primer pairs could amplify 65 polymorphic bands with an average of 3.82 bands per primer pair.The genetic similarity coefficient and cluster analysis were calculated by NTSYS-pc 2.10e software.The genetic analysis showed that,the genetic similarity coefficients between the 10 cultivars ranged from 0.338 to 0.815,and the cluster analysis showed that all the cultivars could be categorized into two major clusters.These results showed that it is an effective and feasible way to develop EST-SSR markers from Asplenium nidus EST sequences,and the primers designed in this study can be used in genetic analysis of fern.

Key words: Asplenium nidus, Molecular marker, EST-SSR

CLC Number:

S682.03

JIA Xin-ping, DENG Yan-ming, SUN Xiao-bo, LIANG Li-jian. Development and Application of EST-SSR Marker in Asplenium nidus[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2015, 30(6): 134-139. doi: 10.7668/hbnxb.2015.06.020.

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