Chung Sang-Min, Staub Jack E
USDA/ARS, Vegetable Crops Unit, Department of Horticulture, 1575 Linden Drive, University of Wisconsin, Madison, WI 53706 USA.
Theor Appl Genet. 2003 Aug;107(4):757-67. doi: 10.1007/s00122-003-1311-3. Epub 2003 Jun 25.
Although universal or consensus chloroplast primers are available, they are limited by their number and genomic distribution. Therefore, a set of consensus chloroplast primer pairs for simple sequence repeats (ccSSRs) analysis was constructed from tobacco (Nicotiana tabacum L.) chloroplast sequences. These were then tested for their general utility in the genetic analysis of a diverse array of plant taxa. In order to increase the number of ccSSRs beyond that previously reported, the target sequences for SSR motifs was set at A or T ( n >/= 7) mononucleotide repeats. Each SSR sequence motif, along with +/-200-bp flanking sequences from the first of each mononucleotide base repeat, was screened for homologies with chloroplast DNA sequences of other plant species in GenBank databases using BLAST search procedures. Twenty three putative marker loci that possessed conserved flanking sequence spans were selected for consensus primer pair construction using commercially available computer algorithms. All primer pairs produced amplicons after PCR employing genomic DNA from members of the Cucurbitaceae (six species) and Solanaceae (four species). Sixteen, 22 and 19 of the initial 23 primer pairs were successively amplified by PCR using template DNA from species of the Apiaceae (two species), Brassicaceae (one species) and Fabaceae (two species), respectively. Twenty of 23 primer pairs were also functional in three monocot species of the Liliaceae [onion (Allium cepa L.) and garlic (Allium sativum L.)], and the Poaceae [oat (Avena sativa L.)]. Sequence analysis of selected ccSSR fragments suggests that ccSSR length and sequence variation could be useful as a tool for investigating the genetic relationships within a genus or closely related taxa (i.e., tribal level). In order to provide for a marker system having significant coverage of the cucumber chloroplast genome, ccSSR primers were strategically "recombined" and named recombined consensus chloroplast primers (RCCP) for PCR analysis. Successful amplification after extended-length PCR of 16 RCCP primer pairs from cucumber ( Cucumis sativus L.) DNA suggested that the amplicons detected are representative of the cucumber chloroplast genome. These RCCP pairs, therefore, could be useful as an initial molecular tool for investigation of traits related to a chloroplast gene(s) in cucumber, and other closely related species.
尽管有通用的或一致的叶绿体引物可供使用，但它们在数量和基因组分布上存在局限性。因此，从烟草（Nicotiana tabacum L.）叶绿体序列构建了一组用于简单序列重复（ccSSR）分析的一致叶绿体引物对。然后对这些引物对在多种植物类群的遗传分析中的通用性进行了测试。为了增加ccSSR的数量，使其超过先前报道的数量，将SSR基序的目标序列设定为A或T（n≥7）单核苷酸重复。使用BLAST搜索程序，在GenBank数据库中筛选每个SSR序列基序以及来自每个单核苷酸碱基重复第一个位置的±200 bp侧翼序列与其他植物物种叶绿体DNA序列的同源性。选择了23个具有保守侧翼序列跨度的推定标记位点，使用市售的计算机算法构建一致引物对。所有引物对在使用葫芦科（6个物种）和茄科（4个物种）成员的基因组DNA进行PCR后均产生了扩增子。最初的23个引物对中，分别有16个、22个和19个通过使用来自伞形科（2个物种）、十字花科（1个物种）和豆科（2个物种）物种的模板DNA进行PCR相继扩增。23个引物对中的20个在百合科的3个单子叶物种[洋葱（Allium cepa L.）和大蒜（Allium sativum L.）]以及禾本科[燕麦（Avena sativa L.）]中也具有功能。对选定的ccSSR片段进行序列分析表明，ccSSR长度和序列变异可作为研究属内或密切相关类群（即族水平）遗传关系的工具。为了提供一个能显著覆盖黄瓜叶绿体基因组的标记系统，对ccSSR引物进行了策略性“重组”，并命名为重组一致叶绿体引物（RCCP）用于PCR分析。从黄瓜（Cucumis sativus L.）DNA进行的16个RCCP引物对的延伸长度PCR后成功扩增表明，检测到的扩增子代表了黄瓜叶绿体基因组。因此，这些RCCP对可作为初步分子工具，用于研究黄瓜及其他密切相关物种中与叶绿体基因相关的性状。
