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Scientia Silvae Sinicae ›› 2015, Vol. 51 ›› Issue (8): 114-120.doi: 10.11707/j.1001-7488.20150815

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Transcriptome Sequencing Analysis and Development of EST-SSR Markers for Pinus koraiensis

Zhang Zhen1,2, Zhang Hanguo1, Mo Chi1, Zhang Lei1   

  1. 1. State Key Laboratory of Tree Genetics and Breeding Northeast Forestry University Harbin 150040;
    2. Research Institute of Subtropical Forestry, CAF Hangzhou 311400
  • Received:2014-10-09 Revised:2014-12-05 Online:2015-08-25 Published:2015-09-10

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Abstract:

[Objective] In Korean pine(Pinus koraiensis) breeding programs, lack of co-dominant genetic markers constrained the development of molecular marker assisted breeding. At present, development of SSR markers based on transcriptome data is still an economic and efficient development strategy of DNA molecular markers. In this study, we used high-throughput sequencing technology to develop EST-SSR markers for Korean pine. Distribution patterns of the markers in the transcriptome sequences and their characteristics were analyzed, in order to provide a basis for analysis of SSR diversity and mutation of Korean pine. [Method] A total of 1 757 SSR sites were identified from 41 476 unigenes in Korean pine transcriptome by using SSR searching program. Statistical analyses were conducted for number, distribution and characteristics of the SSR loci. And 101 pairs of SSR primers were designed and synthesized. Agarose electrophoresis was used for initial check and polyacrylamide gel electrophoresis for separation and detection of the polymorphisms of primers. Then amplification products were collected and sequenced for validation. Finally, 16 pairs of SSR primers and 6 pairs of fluorescence primers were identified. To study the genetic variation, 53 samples of open-pollinated progeny were collected from four seed orchards respectively in Hegang, Linkou, Tieli and Weihe. [Result] The distribution frequency of EST-SSRs (ratio of the number of SSRs to the total number of unigenes) was 4.24%, based on the transcriptome sequences. Mononucleotide dinucleotide and trinucleotide repeats were 46.90%, 17.12% and 34.66% of total SSR, respectively. The SSR repeat number of SSR repeat units was between 5 and 24. Twenty-one pairs of primers showed polymorphism among 101 pairs of primer, which accounting for 20.8% of the total number of primer pairs. By sequencing validation, 16 pairs of primers amplified the target sequence. Eighteen alleles were tested from 6 pairs of fluorescence primers. Polymorphic information content (PIC) was 0.036 3-0.667 4 and the mean was 0.325 0. [Conclusion] Korean pine has a relatively large genome in gymnosperms. The amplified primers of the polymorphism loci were mainly dinucleotide and trinucleotide repeats. The progeny of the Korean pine seed orchard revealed a medium level of polymorphism. This study demonstrated that using Korean pine transcriptome data to develop SSR markers was feasible. The technique of using fluorescent markers to analyze the progeny materials provided a basis for studies of genetic diversity and variation of the Korean pine germplasm resources.

Key words: Pinus koraiensis, transcriptome, EST-SSR, genetic diversity

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