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Development of Juglans Regia SSR Markers by Data Mining of the EST Database

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Abstract

Walnut (Juglans regia), an economically important woody plant, is widely cultivated in temperate regions for its timber and nutritional fruits. Despite abundant studies in germplasm, systemic molecular evaluations of walnut are sparsely reported mainly due to the limited molecular markers available. Expressed sequence tags (EST) provide a valuable resource for developing simple sequence repeat (SSR) markers. In this study, a total of 5,025 walnut ESTs (covering 16.41 Mb) were retrieved from the National Center for Biotechnology Information database. The SSR motifs were then analyzed by the SSRHunter software. In total, 398 SSRs were obtained with an average frequency of 1/4.08 kb. Dinucleotide (di-) repeat motifs accounted for 69.85% of all SSRs, followed by trinucleotide (tri-) with a frequency of 27.64%, while low frequency (2.51%) of tetranucleotide (tetra-) to hexanucleotide (hexa-) was observed. Meanwhile, GCA and TC motifs were prevalent among di- and tri- loci, respectively. Subsequently, a total of 123 primer pairs were designed from the non-redundant SSR-containing unigenes with the selection threshold of SSR length set to 10 bp or more. To examine the efficiency of candidate markers, seven DNA pools were collected from geographically different accessions. Results demonstrated that 41 SSR primer sets could generate high polymorphic amplification products (33.3%), and these polymorphic loci were mainly located in the 3′-untranslated region. Annotation analysis revealed that only two of these 41 loci were located inside open reading frames of characterized proteins (E ≤ 1E−30).

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Acknowledgments

We thank Drs. Chunxian Chen (Citrus Research and Education Center, University of Florida), Zhaocheng Ma, and Qiang Xu (National Key Laboratory of Crop Genetic Improvement) for critically reviewing the manuscript. This research was supported by the doctoral fund of Xinjiang Production and Construction Corp. (2006JC06).

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Authors and Affiliations

  1. Biological Technology Research & Development Center, Tarim University, Alaer, Xinjiang Uygur Autonomous Region, 843300, People’s Republic of China

    Rui Zhang, XinJian Wang, Jun Yu & JiangSheng Gao

  2. National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan, Hubei, 430070, People’s Republic of China

    Rui Zhang, AnDan Zhu, YunJiang Cheng & XiuXin Deng

  3. College of Horticulture and Forestry Science, Huazhong Agricultural University, Wuhan, Hubei, 430070, People’s Republic of China

    Rui Zhang, AnDan Zhu, HongRong Zhang, YunJiang Cheng & XiuXin Deng

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  1. Rui Zhang
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Correspondence to YunJiang Cheng.

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Supplemental Table 1

Polymorphic EST-SSR primer pairs for Juglans regia (DOC 104 kb)

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Zhang, R., Zhu, A., Wang, X. et al. Development of Juglans Regia SSR Markers by Data Mining of the EST Database. Plant Mol Biol Rep 28, 646–653 (2010). https://doi.org/10.1007/s11105-010-0192-2

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