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  

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

作　　者：张增艳[1] 王丽丽[2] 辛志勇[1] 林志珊[1] 

机构地区：[1]中国农业科学院作物育种栽培研究所农业部作物遗传育种重点实验室,北京100081 [2]河北科技大学生物工程与食品科学学院,石家庄050018

出　　处：《Acta Genetica Sinica》2002年第7期627-633,共7页

基　　金：国家"转基因植物专项"(J99 A 0 11);"863"课题 (2 0 0 1AA2 110 11)资助~~

摘　　要：对中间偃麦草麦、小麦和小麦 中间偃麦草 2Ai 2附加系Z1、Z2、Z6 ,代换系ZD2 8等进行RAPD分析 ,从 32 0个RAPD引物中 ,鉴定出 2Ai 2染色体特异的 2个RAPD标记OPO0 5 6 50 和OPM0 4 1 40 0 。利用这 2个特异RAPD引物OPO0 5和OPM0 4 ,PCR扩增普通小麦CS(ABD)及其近缘植物中间偃麦草 (E1 E2 St)、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 ,OPO0 5 6 50 和OPM0 4 1 40 0 均是 2Ai 2染色体上St基因组区域的特异标记。将上述 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(sequencecharacterizedamplifiedregion)标记SC O5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、长穗偃麦草、簇毛麦、黑麦、大麦、粗山羊草以及含有其他中间偃麦草染色体的附加系 ,均没有扩增产物 ,说明上述 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 。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. int

关 键 词：中间偃麦草 染色体特异性 RAPD标记 SCAR标记 St基因组 

分 类 号：Q943.2[生物学—植物学]