粗穗披碱草1H^tS染色体的蛋白质二硫键异构酶基因CAPS标记

Cleaved Amplified Polymorphic Sequence (CAPS) Marker for Protein Disulfide Isomerase Gene Located on Elymus trachycaulus 1H^tS Chromosome

中国春-粗穗披碱草罗伯逊1Ht S.1BL易位系高抗小麦条锈病、叶锈病和白粉病。为了获得抗病小片段易位系,用中国春ph1b基因缺失突变体对1Ht S.1BL进行了诱导,获得了大量诱导后代。本研究用位于小麦1DS染色体的87个EST-STS标记对中国春、粗穗披碱草和1Ht S.1BL易位系进行分析,未发现多态性标记。对其中扩增较好的引物BE444859的PCR产物进行随机克隆测序,获得了14个序列,与已发表的小麦蛋白质二硫键异构酶基因序列具有97%以上的同源性,14个序列均包含3个外显子和2个内含子。限制性酶切位点分析表明,仅2个序列含有HaeIII酶切位点。验证实验结果表明,BE444859/HaeIII组合可以在供试材料中获得多态性。进而用BE444859/HaeIII对一套中国春-粗穗披碱草附加系和151株诱导后代材料进行分析,结果显示,特异多态性条带仅出现在含1Ht S的材料中;68株后代材料均可扩增出多态性带。因此,BE444859/HaeIII可以作为粗穗披碱草蛋白质二硫键异构酶基因CAPS标记,用于检测小麦背景中的1Ht S染色体。本研究揭示当杂交亲本间无直接PCR多态性而又需要建立标记时,发展CAPS标记是行之有效的方法。

Chinese Spring-Elymus trachycauls Robertsonian 1HtS·1BL translocation line is highly resistant to wheat yellow rust,leaf rust and powdery mildew.In order to obtain disease-resistant small chromosome segment translocation,1HtS·1BL was induced by Chinese Spring ph1b gene mutant,and a plenty of offspring are obtained.Development of molecular marker specific for chromosome 1HtS will be helpful for offspring selection.In order to achieve this goal,PCR were performed on Chinese Spring,E.trachycaulus and 1HtS·1BL translocation using 87 pairs of EST-STS primers from EST sequences on wheat 1DS chromosome,however,no polymorphism was found.PCR products from marker BE444859 were randomly cloned and sequenced.As a result,14 sequences,including two introns and there exons each,were obtained.The similarity between these 14 sequences and published wheat protein disulfide isomerase gene sequence is higher than 97%.Verification of PCR using Chinese Spring,E.trachycaulus,1HtS·1BL translocation,a set of Chinese Spring-E.trachycaulus additions and 151 induced offsprings showed that（1） the specific band only existed in material possessing 1HtS chromosome,and（2） the target band was amplified in 68 offsprings.Therefore,BE444859/HaeIII combination could be used as a CAPS marker of protein disulfide isomerase gene for detecting E.trachycaulus 1HtS chromatin in wheat background.The results indicated that development of CAPS marker is useful when no direct PCR polymorphism is present between cross parents.