Stevens M R, Coleman C E, Parkinson S E, Maughan P J, Zhang H-B, Balzotti M R, Kooyman D L, Arumuganathan K, Bonifacio A, Fairbanks D J, Jellen E N, Stevens J J
Department of Plant and Animal Sciences, Brigham Young University, Provo, UT 84602-5157, USA.
Theor Appl Genet. 2006 May;112(8):1593-600. doi: 10.1007/s00122-006-0266-6. Epub 2006 Apr 4.
Quinoa (Chenopodium quinoa Willd.) is adapted to the harsh environments of the Andean Altiplano region. Its seeds have a well-balanced amino acid composition and exceptionally high protein content with respect to human nutrition. Quinoa grain is a staple in the diet of some of the most impoverished people in the world. The plant is an allotetraploid displaying disomic inheritance (2n=4x=36) with a di-haploid genome of 967 Mbp (megabase pair), or 2C=2.01 pg. We constructed two quinoa BAC libraries using BamHI (26,880 clones) and EcoRI (48,000 clones) restriction endonucleases. Cloned inserts in the BamHI library average 113 kb (kilobase) with approximately 2% of the clones lacking inserts, whereas cloned inserts in the EcoRI library average 130 kb and approximately 1% lack inserts. Three plastid genes used as probes of high-density arrayed blots of 73,728 BACs identified approximately 2.8% of the clones as containing plastid DNA inserts. We estimate that the combined quinoa libraries represent at least 9.0 di-haploid nuclear genome equivalents. An average of 12.2 positive clones per probe were identified with 13 quinoa single-copy ESTs as probes of the high-density arrayed blots, suggesting that the estimate of 9.0x coverage of the genome is conservative. Utility of the BAC libraries for gene identification was demonstrated by probing the library with a partial sequence of the 11S globulin seed storage protein gene and identifying multiple positive clones. The presence of the 11S globulin gene in four of the clones was verified by direct comparison with quinoa genomic DNA on a Southern blot. Besides serving as a useful tool for gene identification, the quinoa BAC libraries will be an important resource for physical mapping of the quinoa genome.
藜麦（Chenopodium quinoa Willd.）适应安第斯高原地区的恶劣环境。就人类营养而言，其种子具有均衡的氨基酸组成和极高的蛋白质含量。藜麦谷物是世界上一些最贫困人口饮食中的主食。该植物是异源四倍体，表现出二体遗传（2n = 4x = 36），其二倍体基因组大小为967兆碱基对（Mbp），即2C = 2.01皮克（pg）。我们使用BamHI（26,880个克隆）和EcoRI（48,000个克隆）限制性内切酶构建了两个藜麦细菌人工染色体（BAC）文库。BamHI文库中克隆插入片段平均长度为113千碱基（kb），约2%的克隆没有插入片段；而EcoRI文库中克隆插入片段平均长度为130 kb，约1%没有插入片段。用作73,728个BAC高密度阵列杂交探针的三个质体基因，鉴定出约2.8%的克隆含有质体DNA插入片段。我们估计，合并后的藜麦文库至少代表9.0个二倍体核基因组当量。以13个藜麦单拷贝表达序列标签（EST）作为高密度阵列杂交探针，平均每个探针鉴定出12.2个阳性克隆，这表明基因组9.0倍覆盖率的估计是保守的。通过用11S球蛋白种子贮藏蛋白基因的部分序列探测文库并鉴定多个阳性克隆，证明了BAC文库在基因鉴定中的实用性。通过在Southern杂交中与藜麦基因组DNA直接比较，验证了四个克隆中11S球蛋白基因的存在。除了作为基因鉴定的有用工具外，藜麦BAC文库还将是藜麦基因组物理图谱构建的重要资源。
