PCR-based landmark unique gene (PLUG) markers effectively assign homoeologous wheat genes to A, B and D genomes.
PCR-based landmark unique gene (PLUG) markers effectively assign homoeologous wheat genes to A, B and D genomes.
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基于PCR的地标独特基因（插头）标记有效地将同种小麦基因分配给A，B和D基因组。
DOI：
10.1186/1471-2164-8-135
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发表时间：
2007-05-30
期刊：
影响因子：
4.4
通讯作者：
Nakamura, Toshiki
中科院分区：
文献类型：
--
作者：
Ishikawa, Goro;Yonemaru, Junichi;Saito, Mika;Nakamura, Toshiki
EST-PCR 标记通常代表目标基因的特定产物，因此是遗传分析的有效工具。然而，由于小麦是异源六倍体植物，来自同源基因的PCR产物常常被同时扩增。如果此类产物包含比外显子序列更具多态性的内含子序列，则它们可能更容易区分。然而，小麦的基因组序列数据有限；因此很难预测内含子的位置。利用水稻和小麦基因结构的相似性，我们开发了一种称为PLUG（PCR-based Landmark Unique Gene）的系统来设计引物，使PCR产物包含内含子序列。然后我们研究了使用此类引物扩增的产物是否可以作为能够区分源自同源基因的多种产物的标记。 PLUG系统由以下步骤组成：（1）提取与小麦UniGene序列具有高度同源性的单拷贝水稻基因（Landmark Unique Gene loci；LUG）； (2)利用LUG与小麦UniGene序列进行比对分析，预测外显子-外显子连接，提取可用于设计小麦内含子侧翼引物(TaEST-LUG)的LUG； (3)以交互方式设计引物。从总共 4,312 个 TaEST-LUG 中，随机选择 24 个位点并用于设计引物。利用所有这些引物组，我们从靶基因获得了特异性的、含有内含子的产物。使用小麦无效体-四体系将这些标记分配给染色体。通过使用琼脂糖凝胶电泳的 PCR-RFLP 分析，24 个标记中的 19 个位于至少一条染色体上。在开发能够有效分选同源基因衍生产物的小麦EST-PCR标记时，设计能够扩增包含具有插入/缺失多态性的内含子序列的产物的引物非常重要。使用PLUG系统，根据与水稻的比较基因组学选择可用于标记开发的小麦EST序列，然后以交互式、半自动的方式制备内含子序列侧翼的引物组。因此，PLUG系统是大规模标记开发的有效工具。
EST-PCR markers normally represent specific products from target genes, and are therefore effective tools for genetic analysis. However, because wheat is an allohexaploid plant, PCR products derived from homoeologous genes are often simultaneously amplified. Such products may be easier to differentiate if they include intron sequences, which are more polymorphic than exon sequences. However, genomic sequence data for wheat are limited; therefore it is difficult to predict the location of introns. By using the similarities in gene structures between rice and wheat, we developed a system called PLUG (PCR-based Landmark Unique Gene) to design primers so that PCR products include intron sequences. We then investigated whether products amplified using such primers could serve as markers able to distinguish multiple products derived from homoeologous genes. The PLUG system consists of the following steps: (1) Single-copy rice genes (Landmark Unique Gene loci; LUGs) exhibiting high degrees of homology to wheat UniGene sequences are extracted; (2) Alignment analysis is carried out using the LUGs and wheat UniGene sequences to predict exon-exon junctions, and LUGs which can be used to design wheat primers flanking introns (TaEST-LUGs) are extracted; and (3) Primers are designed in an interactive manner. From a total of 4,312 TaEST-LUGs, 24 loci were randomly selected and used to design primers. With all of these primer sets, we obtained specific, intron-containing products from the target genes. These markers were assigned to chromosomes using wheat nullisomic-tetrasomic lines. By PCR-RFLP analysis using agarose gel electrophoresis, 19 of the 24 markers were located on at least one chromosome. In the development of wheat EST-PCR markers capable of efficiently sorting products derived from homoeologous genes, it is important to design primers able to amplify products that include intron sequences with insertion/deletion polymorphisms. Using the PLUG system, wheat EST sequences that can be used for marker development are selected based on comparative genomics with rice, and then primer sets flanking intron sequences are prepared in an interactive, semi-automatic manner. Hence, the PLUG system is an effective tool for large-scale marker development.