小麦背景下冰草6P染色体特异EST标记的开发

Development of EST Markers Specific to Agropyron cristatum Chromosome 6P in Common Wheat Background

小麦-冰草[Agropyroncristatum(L.)Gaertn.]6P附加系普冰4844-12具有多粒等可用于小麦改良的优异基因。为高效率检测由小麦-冰草6P附加系衍生的易位系和渐渗系,以普冰4844-12及其亲本普通小麦Fukuhokomugi和冰草Z559(2n=4x=28,PPPP)为材料,通过对冰草转录组测序获得的EST序列设计的P基因组EST分子标记引物,筛选在普通小麦背景下的6P染色体特异分子标记。结果从1453对P基因组EST引物中筛选出130对小麦-冰草6P附加系特异标记引物。进而将这些特异标记与NCBInr蛋白质数据库及小麦EST序列进行了比对,发现4条冰草EST序列的功能注释与抗病、抗逆相关;36条冰草EST与已经定位的小麦EST具有较高的相似性,其中33条(91.67%)位于小麦第6部分同源群染色体。为了进一步验证这些标记的特异性,分别对其中4个具有功能注释的EST标记在中国春等7个普通小麦背景下和随机选择的5个标记在小麦-冰草6P易位系背景下进行了检测,结果证明其确实可用于检测6P染色体。这些冰草6P染色体特异标记的开发为大规模地鉴定小麦-冰草衍生后代中P染色质成分奠定了基础。

Wheat-Agropyron cristatum （L.） Gaertn. 6P addition line has the characteristics of superior numbers of florets and kernels per spike, which can be used in the improvement of wheat varieties. In this study, we developed EST markers specific to the A. cristatum 6P chromosome using the wheat-A. cristatum 6P disomic addition line 4844-12 （2n=44） and its common wheat parent ＂Fukuhokomugi＂ as well as A. cristatum accession Z559 （2n=4x=28, PPPP）. PCR primers were designed according to the sequences of ESTs gained by the transcriptome sequencing of A. cristatum. A total of 130 6P-specific molecular markers were developed from 1 453 PCR primers. These specific markers were matched with protein database from NCBI and wheat EST sequence to understand the potential function genes on chromosome 6P and the gene synteny between A. cristatum and common wheat. The results showed that functions of four A. cristatum EST sequences are related to disease and stress resistance. Thirty six A. cristatum EST sequences could be matched with the located wheat EST bin map, in which 33 （91.67%） sequences were located in the sixth homoeologous group. To validate the specificity of these markers, we detected four EST specific markers with functional annotation under different backgrounds of other seven wheat varieties. Five specific markers were selected randomly to detect wheat-A. cristatum 6P translocation lines. The results showed that these specific markers can be used to detect chromosome 6P of A. cristatum in common wheat background.