【摘要】 【目的】探究从前苏联引进的簇毛麦No.1026(Dv#4)的EST-SSR序列特征及其在染色体的分布,分析它们在不同簇毛麦间及与小麦间的多态性,为其进一步的研究与利用提供依据。【方法】通过Illumina HiSeq测序获得No.1026植株的转录组序列,利用MISA软件分析转录组SSR序列及特征,采用Primer 3设计SSR引物,随机合成238对引物,对小麦中国春与簇毛麦Dv#4和引自英国剑桥的簇毛麦Dv#2的基因组DNA进行扩增,在琼脂糖凝胶上分离评价扩增产物的多态性,并利用一套小麦-簇毛麦异染色体系进行扩增分析。【结果】检测了No.1026总长62.76 Mb的转录组序列,发现10 497个SSR位点,它们分布于8 735条Unigene上。在1—6个核苷酸的重复单元中,单、双、三碱基的重复占95.85%,其中三核苷酸串联重复数量最多,占SSR总数的50.33%,而CCG/CGG基序的重复占三核苷酸串联重复的41.66%;单核苷酸重复是第二大类型,出现频率为27.13%,其中A/T重复占单核苷酸重复的74.58%。二核苷酸重复类型的数量位列第三,占SSR总数的18.39%。在238对EST-SSR引物中,88对在中国春与簇毛麦(包括Dv#2和Dv#4)之间的扩增产物显示多态性;8对只在簇毛麦和单一异染色体系扩增;4对可在簇毛麦和多个异染色体系中特异扩增;但多数可在簇毛麦中清晰扩增的引物不能在任何异染色体系中扩增,推测可能与簇毛麦基因组及染色体导入小麦过程中的变异有关;47对(19.74%)在2份不同来源的簇毛麦Dv#2和Dv#4之间的扩增产物呈现多态性。利用1对EST-SSR引物和1个EST-PCR标记检测48个簇毛麦植株,证明多态性的SSR引物可有效用于检测簇毛麦的异质性。【结论】簇毛麦No.1026(Dv#4)的转录组中存在丰富的SSR序列,其中CCG/CGG、A/T和AG/CT等三核苷酸、单核苷酸和二核苷酸是其最主要的串联重复基序。部分EST-SSR的侧翼保守序列与单一或若干外源染色体特异相关联,据此开发特异的分子标记,可用于跟踪检测小麦背景中特异的簇毛麦染色体或染色体片段。Dv#4与Dv#2间的部分EST-SSR具有多态性,提示2份不同来源簇毛麦的表达序列间存在一定程度的遗传差异,因此,簇毛麦Dv#4值得进一步的发掘研究。更多还原

【Abstract】 【Objective】The aim of this study is to explore the characteristics of the EST-SSR sequences of a Dasypyrum villosum accession No.1026(Dv#4) introduced from the former Soviet Union, and their distributions on chromosomes and polymorphism in different D. villosum accessions and between Dv#4 and common wheat. 【Method】Transcriptome sequences of Dv#4 plants were obtained by Illumina HiSeq sequencing and used to search and analyze the SSR sequences using MISA software and design primers by Primer 3. In total, 238 pairs of primers were selected randomly for synthesis and used to amplify the genomic DNAs of wheat Chinese Spring(CS) and the two different D. villosum accessions. The polymorphisms of the PCR products on agarose gel were evaluated. Further, the features of their chromosome distributions in Dv#4 were studied by using a set of wheat-D. villosum alien chromosome lines(including disomic addition lines and disomic substitution lines). 【Result】 A total sequence length of 62.76 Mb was detected and 10 497 SSR loci were found in the transcriptome data. They are involved in 8 735 unigenes. Repeated unit of mono-, di-, tri-nucleotides are the main type, holding 95.85% of all loci among 1-6 nucleotides repeats, among which tri-nucleotide is the richest component that makes up of 50.33% and contains CCG/CGG motif by 41.66%. The next component is mono-nucleotide tandem repeat, and its occurrence frequency is 18.39%, and A/T repeats occupy 74.58% in this type. Di-nucleotide ranks the 3 rd, and it holds 18.39% in the total SSR loci. Among 238 pairs of randomly synthetized EST-SSR primers, 88 pairs amplified polymorphic fragments between CS and D. villosum(including Dv#2 and Dv#4); 8 pairs only had amplifications in D. villosum and some single alien chromosome lines; 4 pairs could specifically amplify bands in D. villosum and multiple alien chromosome lines. But, many primers which had amplification in both D. villosum accessions had no amplification in any alien chromosome lines. Therefore, it can be inferred that variations on the flanking conserved sequences of SSR might occur during the transferring of the exogenous genome or chromosomes into wheat. Additionally, 47 pairs of primers(19.74%) showed polymorphisms between Dv#2 and Dv#4. By using a pair of EST-SSR primer and a PCR marker, respectively, polymorphic amplicons were detected in 48 D. villosum plants, indicating that polymorphic SSR primer can be used to detect the heterogeneity of D. villosum effectively.【Conclusion】 D. villosum Dv#4 has abundant SSR sequences in its transcriptome, of which, CCG/CGG, A/T, AG/CT and other tri-, mono-, and di-nucleotide are the main tandem repeats. The flanking conserved sequences of partial EST-SSR in Dv#4 are associated with a single or several chromosomes specifically, which provides an ideal sequence resource for the development of specific molecular markers to track and detect D. villosum chromosomes or chromosome fragments in wheat background. Some of the EST-SSR polymorphism between Dv#4 and Dv#2 indicates that there is a certain degree of genetic diversity in some of the expressed sequences between the two different origin D. villosum accessions. Therefore, Dv#4 is valuable to be further investigated.更多还原