功能型分子标记(ISAP)的开发及评价

Development and appraisement of functional molecular marker: intron sequence amplified polymorphism (ISAP)

分子标记在图谱构建,QTL分析,基因定位以及标记辅助育种中起着越来越重要的作用。研究者都期望一个分子标记位点代表一个特定的基因,甚至与某种性状联系起来,这样,通过对某个分子标记的筛选即能对性状进行筛选,此即功能型分子标记。而目前广泛使用的基于PCR基础的分子标记如RAPD、SSR、AFLP等或是扩增非编码区域,或是随机在基因组中扩增,得到的位点一般与目标性状基因距离较远,这使得分子标记在应用上与其目标有一定的偏差。研究建立了一种基于基因中内含子序列的功能型分子标记,试图使标记位点与基因序列联系起来以达到其功能型的目的。它利用内含子剪接位置的保守一致序列作为其引物的核心序列,其上下游引物均为18bp,上下游引物间通过组合配对的方式作为扩增的引物对,对真核生物的基因序列进行扩增。为了验证ISAP的功能性,研究设计了17条引物(9条上游引物,8条下游引物,共计72个引物组合)对棉花F2群体进行扩增并构建遗传连锁图谱,其中67个显示了多态性,共得到212个位点。我们用此212个位点连同164个SRAP位点构建了一张包含276个位点的遗传连锁图谱,ISAP标记在整个连锁群中分布比较均匀,部分区域呈现标记高饱和现象,可能为编码序列富集区。另外对20个片段进行测序的结果表明,85%的序列显示了与已公布EST序列的同源性,说明扩增是跨越了外显子进行的,得到的序列与表达序列紧密连锁。结果显示,ISAP标记是简单,可靠,具有较高多态性,并且扩增基因区域的一种功能型分子标记。同时,还使用ISAP标记对其他植物进行了扩增,取得了良好的效果。

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 application 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 × 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 se- quences. These results showed that ISAP marker system was simple, efficient, reliable, and had a relatively high polymor- phism, 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.