Scientia Agricultura Sinica ›› 2013, Vol. 46 ›› Issue (1): 9-17.doi: 10.3864/j.issn.0578-1752.2013.01.002

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

Three-Dimensional Pooling and HvGW2 Gene Screening of Barley (Hordeum vulgare L.) BAC Library

 GUO  Gang-Gang, DONG  Guo-Qing, ZHOU  Jin, DA  Wa-顿Zhu, YUAN  Xing-Miao, ZHANG  Jing   

  1. 1.Institute of Crop Science, Chinese Academy of Agricultural Sciences/Key Laboratory of Crop Germplasm and Biotechnology, Ministry of Agriculture/National Key Facility for Crop Gene Resources and Genetic Improvement, Beijing 100081
    2.Wuhan Polytechnic University, Wuhan 430023
    3.Tibet Academy of Agricultural and Animal Husbandry Sciences, Lhasa 850032
  • Received:2012-10-12 Online:2013-01-01 Published:2012-11-29

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Abstract: 【Objective】 The objective of this study was to isolate HvGW2 gene from barley BAC library rapidly. 【Method】 Three-dimensional (3D) BAC pools were constructed and screened by using barley GW2 gene-specific PCR primers. Positive BAC clones were screened out and further sequenced, the obtained HvGW2 gene was used for homologous evolutionary analysis with those of other grasses. 【Result】 One hundred and sixty BAC DNA pools were prepared by 3D pooling strategy from barley BAC library with only 0.18% of empty clones. PCR-based screening demonstrated that only one of the three positive BAC clones (196M02) contained the complete coding sequence of HvGW2. Sequence analysis showed that barley GW2 gene contains eight exons and seven introns, which revealed that it has similar gene structure and conserved functional domain with other plant species. Furthermore, HvGW2 has the highest homologous with wheat GW2 protein. Moreover, the sixth intron of HvGW2 gene contains two different retrotransposon insertions, which resulted in a 11.5 kb length increase of barley GW2 gene than the other crops.【Conclusion】 By constructing three-dimensional BAC pools, and combining with PCR-based screening, positive BAC clones which contain target gene can be isolated quickly. Not only the quality of the barley BAC library was evaluated, but also an auxiliary platform for barley gene cloning was established. It was speculated that the retrotransposon insertions in gene intron might be the key variation which affected the gene transcription levels of HvGW2.

Key words: barley , BAC library , three-dimensional pools , HvGW2 , grain weight gene

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