中间偃麦草染色体2Ai-2特异PCR新标记的建立和St基因组特异序列的克隆

中间偃麦草麦、小麦和小麦－中间偃麦草2Ai－2附加系Z1、Z2、X6，代换系ZD28等进行RAPD分析，从320个RAPD引物中，鉴定出2Ai－2染色体特异的2个RAPD标记OPO05650和OPMO414000。利用这2个特异OPO05和OPM04，PCR扩增普通小麦CS（ABD）及其近缘植物中间偃麦草（E1E2St）、拟鹅冠草（St），长穗偃麦草（E）、簇毛麦（V）、黑麦（R）、大麦（H）粗山羊草（D）等基因组DNA。结果表明，OPO05650和OPO41400均是2Ai－2染色体上St基因组区域的特异标记。将上棕2个特异片段分离回收、克隆、测序，根据测序结果重新设计、合成特异引物，成功地转换RAPD标记为SCAR（sequence characterizked amplifed region）标记SC－05和SC－M4。利用SCAR标记对不同材料进行分析的结果表明，凡含有2Ai－2染色体的抗黄矮病材料及拟鹅冠草均产生一条扩增带，不含2Ai－2染色体的材料，包括小麦、长穗麦草、簇毛麦、黑麦、在麦、粗山羊草以有含有其他他中间偃麦草染色休的附加系，均没有扩增产物，说明上棕2个SCAR标记是中间偃麦草2Ai－2染色体的特异性PCR标记，且是2Ai－2染色体上St基因组区域的特异性标记。克隆与鉴定中间偃麦草的2个SCAR扩增片段TiSCO5和TiSCM4。结果表明，克隆的中间偃麦草TiSCO5和TiSCM4特异片段，分别是St基因组特异性的寡拷贝序列有多拷贝重复序列，为St基因组遗传研究的新探针。

Development of New PCR Markers Specific to a Thinopyrum intermedium Chromosome 2Ai-2 and Cloning of the St-specific Sequences

To meet the need of selecting translocation lines, some new RAPD markers for 2Ai 2 chromosome of Th. intermedium were identified in the paper. Out of 320 RAPD primers, 2 specific primers,OPO05 and OPM04,can amplify respectively a specific band with size of about 650bp and 1400bp in the BYDV resistant materials containing the chromosome 2Ai 2,including Th. intermedium, wheat Th. intermedium partial amphipoild Zhong 4 Awnless, addition lines Z1, Z2 and Z6, F 1 (Z2/Wan7107) and substitute line ZD28 etc, but absent in all the materials lacking the 2Ai 2 chromosome , including susceptible wheat parents and other wheat Th. intermedium addition lines L1 and Z4. The RAPD markers specific to chromosome 2Ai 2, OPO05 650 and OPM04 1400 , may be located on the St genomic region of 2Ai 2 chromosome by PCR analysis on Th. intermedium (E 1E 2St), Pseuderogneria strigosa (St), Th. elongatum (E), Haynaldia villosa (V), Se cale cereale (R), Hordeum vulgare (H), Aegilops squrrosa (D) and Triticum aestivum (ABD) genomic DNA. The specific bands of RAPD markers OPO05 650 and OPM04 1400 were isolated and cloned. After the clones were subjected to restrict digestion analysis,PCR and Southern hybridization analysis, some clones were sequenced. Based on the sequences, 1 pair of primers SC O5(U+L) and 1 pair of primers SC M4(U+L) were designed, synthesized and used to amplify the materials with and without 2Ai 2 chromosome. The results showed that the SCAR markers of chromosome 2Ai 2, SC O5 and SC M4, were converted successfully from the RAPD markers OPO05 650 and OPM04 1400 . The Th. intermedium fragments amplified by the primers of SC O5 (U+L) and SCM4(U+L) were cloned and analyzed. The results of Southern hybridization indicated that TiSCO5, the cloned fragment of Th. intermedium amplified by primers of SC O5(U+L) was a low copy sequence specific to St genome, and another sequence TiSCM4, the cloned fragment of Th. intermedium amplified by primers of SC M4(U+L) was a repeat sequence specific to St genomic. The sequences will be new probes to detect St genomic chromatin.