A deep-coverage BAC library for Ponkan mandarin and its further use in screening ethylene regulated genes
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- 作者：Miao Wang ; AnQun Tan ; LingYun Zhang ; JiaWei Yang ; ZhaoXin Wei ; ChunZhen Cheng ; ZhiQin Zhou ; GuangYan Zhong
- 关键词：Citrus ; Bacterial artificial chromosome (BAC) library ; Ethylene regulated genes ; PCR screening
- 刊名：Scientia Horticulturae
- 出版年：2010
- 出版时间：22 November 2010
- 年：2010
- 卷：127
- 期：1
- 页码：64-69
- 全文大小：321 K
文摘
A deep-coverage bacterial artificial chromosome (BAC) library of Yanxiwanlu Ponkan, a late maturing cultivar, was constructed and evaluated. Results showed that the library contained a total of 61,000 clones. Restriction analysis indicated that the DNA insert sizes ranged from 30 to 170 kb with an average of 82 kb. No empty clone was found. Therefore, the library should cover 13.6-fold of the citrus genome. Continuous sub-cultivation of 5 randomly chosen clones for up to 100 generations showed no detectable change in their restriction profiles. To demonstrate the application of the library in discovery of genes, 7 ethylene-regulated genes were screened by PCR amplification from hierarchically pooled library clones with gene-specific primers. Five positive clones, each representing a different gene, were thus identified from 3840 clones. Sequencing of the clones showed that all of them matched the expected genes, indicating that the library was highly representative.