Lu Cai-Rui, Yu Shu-Xun, Yu Ji-Wen, Fan Shu-Li, Song Mei-Zhen, Wang Wu, Ma Shu-Juan
China Cotton Research Institute, Key Laboratory of Cotton Genetic Improvement of Ministry of Agriculture of China, Anyang 455004, China.
Yi Chuan. 2008 Sep;30(9):1207-16. doi: 10.3724/sp.j.1005.2008.01207.
Molecular markers are playing an increasingly important role in map construction, QTL analysis, gene mapping and marker-assisted selection. Researchers hope the target gene and locus are as close as possible, one locus can present one gene, or linked with some important trait, then, individuals with useful trait can be selected through molecular markers selecting, and it's the functional molecular marker. PCR-based molecular markers such as RAPD, SSR, AFLP amplified non-coding regions, or the whole genome randomly, the locus is far away from the gene of targeted trait, this limit the ap-plication of these molecular markers. This study established a kind of functional molecular markers based on intron of gene sequence, trying to link loci with gene sequence to achieve the purpose of its function. It used the conservative consistent sequence of intron splicing sites as its core sequence of amplification. ISAP is a PCR-based marker system, it has two kinds of primers: forward primer and reverse primer, both primers are 18 bases. Any of the primers can be used to construct a primer combination with the other kind of primers. Seventeen primers, 9 forward and 8 reverse, were used to construct 72 primer combinations, 67 of them showed polymorphism in a G. hirsutum cv. CCRI36 x G. barbadense cv. H7124 F2 population and a total of 212 loci were obtained. Together with 164 SRAP loci, these 212 loci were used to construct a genetic linkage map. ISAP markers distributed evenly in the entire linkage group, part of the region had a high saturation, might be the coding sequence-rich region. Sequencing results of 20 fragments showed that 85% of the sequences announced homology with published EST sequence stored in the NCBI which indicated that they were amplified adjacent to expressed sequences. These results showed that ISAP marker system was simple, efficient, reliable, and had a relatively high polymorphism, furthermore, it directly targeted gene sequence, was a functional molecular marker system. ISAP was also used to amplify other plants and good results were achieved.
分子标记在图谱构建、QTL分析、基因定位和标记辅助选择中发挥着越来越重要的作用。研究人员希望目标基因和位点尽可能接近，一个位点能代表一个基因，或与某些重要性状连锁，然后通过分子标记选择来挑选具有有用性状的个体，这就是功能分子标记。基于PCR的分子标记如RAPD、SSR、AFLP随机扩增非编码区或整个基因组，位点与目标性状的基因距离较远，这限制了这些分子标记的应用。本研究基于基因序列的内含子建立了一种功能分子标记，试图将位点与基因序列联系起来以实现其功能目的。它以内含子剪接位点的保守一致序列作为扩增的核心序列。ISAP是一种基于PCR的标记系统，它有两种引物：正向引物和反向引物，两种引物均为18个碱基。任何一种引物都可与另一种引物构建引物组合。使用17条引物（9条正向引物和8条反向引物）构建了72个引物组合，其中67个在陆地棉品种CCRI36×海岛棉品种H7124的F2群体中表现出多态性，共获得212个位点。将这212个位点与164个SRAP位点一起用于构建遗传连锁图谱。ISAP标记均匀分布在整个连锁群中，部分区域具有较高饱和度，可能是富含编码序列的区域。20个片段的测序结果表明，85%的序列与NCBI中存储的已发表EST序列具有同源性，这表明它们是在表达序列附近扩增得到的。这些结果表明，ISAP标记系统简单、高效、可靠，具有较高的多态性，此外，它直接针对基因序列，是一种功能分子标记系统。ISAP也被用于扩增其他植物并取得了良好效果。
