麦类作物原位杂交影响因素的研究

INFLUENCE FACTORS OF IN SITU HYBRIDIZATION IN TRITICEAE

为了更好地利用原位杂交技术进行麦类作物外源染色体的检测和基因定位，对麦类作物原位杂交的影响因素进行了研究。（１）利用缺口转译法对克隆的ＤＮＡ 片段进行生物素标记，即节省时间，又可以获得较高的标记效率；对麦类作物的基因组ＤＮＡ，宜采用随机寡核苷酸引物标记法进行生物素标记，适当延长标记时间可以提高标记效率。（２）共变性法比较适宜麦类作物的原位杂交，分别变性法如掌握不当易使染色体产生膨胀现象，变性温度过高也会使黑麦（Ｓｅｃａｌｅ ｃｅｒｅａｌ）染色体的轮廓模糊不清。（３）应根据麦类作物亲本之间的亲缘关系决定封阻ＤＮＡ 的使用浓度，并利用生物素标记的簇毛麦（Ｈａｙｎａｌｄｉａ ｖｉｌｌｏｓａ）基因组ＤＮＡ 为探针，从普通小麦（Ｔｒｉｔｉｃｕｍ ａｅｓｔｉｖｕｍ ）－簇毛麦双二倍体的染色体中识别了簇毛麦的染色体。（４）麦类作物的原位杂交受洗脱强度的影响很大，利用甲酰胺进行洗脱可以获得背景清晰的原位杂交带型。随着原位杂交技术分辨率的不断提高。

In order to increase the efficiency, accuracy, fidelity and reliability of in situ hybridization to identify the alien chromosomes and chromosome fragments in triticeae, major steps including probe labelling, chromosome denaturation, DNA concentration for blocking and post hybridization washing in in situ hybridization were optimized. The results are as fellows. (1) The cloned repetitive DNA sequence could be biotin labelled more efficiently by nick translation than by random oligonucleotide labelling method; whereas the random oligonucleotide labelling is more suitable for genomic DNA probe and the labelling efficiency could be increased by prolonging the labelling time appropriately. (2) Denaturation of the biotinylated probe and chromosomes together in oven at 75℃ showed the satisfactory results of in situ hybridization, but the contour of treated rye chromosomes often became blurred when the temperature of denaturation was higher than 85℃. When 70% formamide (in 2×SSC) was used to denature the chromosome DNA, rye chromosomes often swelled although the biotinylated signals could be detected. (3) The unlabeled DNA concentrations for blocking were tested in genomic in situ hybridization to detect the Haynaldia villosa chromosomes with biotin labelled H. villosa genomic DNA as probe. The best contrast between H. villosa and wheat chromosomes was obtained without using the blocking DNA (unlabeled wheat genomic DNA). (4) Post hybridization washes were carried out in 50% formamide (in 2×SSC) or in 2×SSC at different temperature. When the post hybridization washing temperature were increased gradually from room temperature to 42℃ in 50% formamide (in 2×SSC), specific in situ hybridization signals on chromosome in triticeae were observed using both biotinylated repetitive DNA and genomic DNA as probe. With the improved resolution of this protocol, in situ hybridization would be widely applied to wheat breeding and genetics researches.