Construction of BAC and BIBAC libraries from sunflower and identification of linkage group-specific clones by overgo hybridization
Construction of BAC and BIBAC libraries from sunflower and identification of linkage group-specific clones by overgo hybridization
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DOI：
10.1007/s00122-006-0265-7
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发表时间：
2006-06-01
影响因子：
5.4
通讯作者：
Jan, C. C.
中科院分区：
文献类型：
--
作者：
Feng, Jiuhuan;Vick, Brady A.;Jan, C. C.
利用向日葵品种ha89的核DNA构建了互补BAC和BIBAC文库。在pECBAC1载体上用BamHI构建的BAC文库包含107,136个克隆，平均插入大小为140 kb。BIBAC文库以HindIII为载体，构建于具有植物转化能力的二进制载体pCLD04541中，包含84,864个克隆，平均插入大小为137 kb。这两个文库总共包含192,000个克隆，相当于大约8.9个向日葵单倍体基因组（3,000 Mb/1C），并且提供大于99%的可能性获得感兴趣的克隆。估计BAC和BIBAC克隆携带叶绿体或线粒体DNA序列的频率分别为2.35%和0.04%，插入空克隆的频率小于0.5%。为了便于染色体工程和将向日葵遗传图谱锚定在其染色体上，利用向日葵每个连锁群中的1 - 3个单拷贝或低拷贝RFLP标记设计重叠寡核苷酸对（overgos）。设计了36个overgos，并将其汇集为探针，以筛选BAC和BIBAC库的一个子集（5.1倍）。在这36例中，33例（92%）至少产生了一个阳性克隆，3例（8%）没有击中任何克隆。结果表明，共鉴定出195个BAC和BIBAC克隆，代表19个连锁群，其中BAC克隆76个，BIBAC克隆119个，进一步验证了该文库的基因组覆盖率和实用性。这些BAC和BIBAC文库以及连锁群特异性克隆为向日葵基因组的全面研究提供了必要的资源。
Complementary BAC and BIBAC libraries were constructed from nuclear DNA of sunflower cultivar HA 89. The BAC library, constructed with BamHI in the pECBAC1 vector, contains 107,136 clones and has an average insert size of 140 kb. The BIBAC library was constructed with HindIII in the plant-transformation-competent binary vector pCLD04541 and contains 84,864 clones, with an average insert size of 137 kb. The two libraries combined contain 192,000 clones and are equivalent to approximately 8.9 haploid genomes of sunflower (3,000 Mb/1C), and provide a greater than 99% probability of obtaining a clone of interest. The frequencies of BAC and BIBAC clones carrying chloroplast or mitochondrial DNA sequences were estimated to be 2.35 and 0.04%, respectively, and insert-empty clones were less than 0.5%. To facilitate chromosome engineering and anchor the sunflower genetic map to its chromosomes, one to three single- or low-copy RFLP markers from each linkage group of sunflower were used to design pairs of overlapping oligonucleotides (overgos). Thirty-six overgos were designed and pooled as probes to screen a subset (5.1x) of the BAC and BIBAC libraries. Of the 36 overgos, 33 (92%) gave at least one positive clone and 3 (8%) failed to hit any clone. As a result, 195 BAC and BIBAC clones representing 19 linkage groups were identified, including 76 BAC clones and 119 BIBAC clones, further verifying the genome coverage and utility of the libraries. These BAC and BIBAC libraries and linkage group-specific clones provide resources essential for comprehensive research of the sunflower genome.