摘要
为在烟草(Nicotiana tabacum)上进行IMP(inter-MITE polymorphism,MITE位点间多态性)标记开发,利用美国烟草基因组测序数据和MITE(miniature inverted repeat transposable element,微型反向重复转座元件)结构特征,通过生物信息学方法获得11 581组具有特异TIR(terminal inverted repeat,末端重复元件)序列的MITE及其多序列联配结果,并根据联配结果设计219条烟草IMP引物;利用9个不同类型烟草代表性品种进行测试,其中119条引物PCR扩增成功,引物开发成功率为54.6%;进一步对一个烤烟遗传群体亲本‘红花大金元’和‘HicksBroadleaf’进行标记筛选,共获得29个多态性标记,群体检测表明有21个标记可以整合定位到一个SSR标记遗传连锁图上.说明IMP标记在烟草中是一种可以利用的分子标记,但跟其他标记一样表现出较低的多态性,需高通量检测平台与之配套来提高其开发效率.
DNA transposable elements require the transposase from an autonomous parent element for transposition. The characteristic of MITEs are generally less than 500 bp with terminal inverted repeat （TIR） and target site duplication （TSD）. MITEs often insert into insides of genes or regions near to genes, and therefore the markers based on TIRs of MITEs termed inter-MITE polymorphism （IMP）, are possibly lined to important agricultural traits and have been successfully used in barley, wheat, maize, oats etc. Extremely low polymorphism has been observed in tobacco by several molecular marker systems, such as RAPD, AFLP, ISSR, SRAP and SSR, due to its narrow genetic background during domestication. In this study, IMP markers were developed in tobacco using genomic sequences from the Tobacco Genome Initiative （TGI）. Non-redundant tobacco genomic sequences from TGI were used to scan MITEs through a bioinformatic approach and 11 581 MITEs of unique TIRs were eventually identified and aligned （partial result was shown in Fig. 2）. Two hundred and nineteen IMP primers were designed according to the alignments and then were tested among nine representative tobacco varieties with different types （flue-cured, burley, oriental and cigar tobacco） （Table 1）. In the 219 primers, 119 were successfully amplified by PCR, which demonstrated a success rate of 54.6 % （partial result was shown in Fig. 3）. The 119 primers were further used in a doubled haploid （DH） population of a flue-cured tobacco, 29 of which showed polymorphism between the two parental lines （Honghuadajinyuan/Hicks Broadleaf）. Of the 29, 21 were genotyped in the DH population and integrated into seven linkage groups with SSR markers （Fig. 5）. Our results demonstrate that IMP markers are useful in tobacco such as genetic germplasm investigation and genetic map construction. But low development efficiency is also observed for this kind of marker system in tobacco. To improve the efficiency of IMP marker development, high-throughp
出处
《浙江大学学报（农业与生命科学版）》
CAS
CSCD
北大核心
2012年第6期655-661,共7页
Journal of Zhejiang University:Agriculture and Life Sciences
基金
云南省科技厅资助项目(2008CD194)
云南省烟草公司资助项目(08A05)
