基于TRAP的长穗偃麦草SCAR标记的开发及应用

Development and Applications of SCAR Markers Specific to Thinopyrum elongatum by TRAP Technology

为了开发长穗偃麦草的特异标记,依据TRAP技术,利用56对固定引物与随机引物的组合对中国春-长穗偃麦草附加系等材料进行PCR扩增,共筛选出160条分布于长穗偃麦草1E-7E染色体的特异扩增片段。通过对104个片段的序列进行同源比对,选择与小麦无同源序列的区段设计138对引物,分别对中国春、长穗偃麦草、中国春-长穗偃麦草附加系进行PCR扩增,最终获得30个长穗偃麦草特异SCAR标记,发展标记的效率为53.6%。利用这些特异SCAR标记对硬粒小麦(AABB)与异源六倍体小麦(AABBEE)杂交产生的F2中38个单株进行扩增鉴定,9个单株仅附加了1条相同的E染色体,其余为多条E染色体或者无E染色体附加。这些SCAR标记在不同材料和世代表现出优越的特异性和稳定性,可用于检测小麦背景中附加的相关长穗偃麦草染色体。

Thinopyrum elongatumis an important alien gene pool for wheat genetic improvement.It is necessary to develop specific markers of Th.elongatum for its useful genes to be transferred into wheat.Fifty six pairs of fixed and arbitrary primers were used in this study.One hundred and sixty amplified fragments from Th.elongatum chromosomes 1Eto 7E were obtained.Among them,104 fragments were sequenced and compared with the existing sequence of wheat in GenBank.One hundred and thirty eight pairs of PCR primers were redesigned based on results of sequence alignment analysis.DNAs of Chinese Spring,Th.elongatum and series of wheat-Th.elongatum addition lines were amplified by 138 pairs of redesigned specific primer.Eventually,thirty SCAR markers specific to Th.elongatum were developed with a successful rate of 53.5% and each E-chromosomes of Th.elongatumcould be identified using these SCAR markers.These specific SCAR markers were used to screen 38F2 plants derived from the cross of a durum 'Langdon'(AABB)with synthesized hexaploid wheat(AABBEE).The results indicated that 9plants had an E chromosome addition;the rest waswith more E chromosomes or no E chromosome addition among the 38F2 plants.The results showed that these specific SCAR markers exhibited excellent specificity and stability not only in different E chromosomes-involved materials,but also in different generations.Therefore,these specific markers can be used for detection and tracking of Th.elongatumchromosomes in wheat background.