基于簇毛麦No.1026转录组的SSR序列分析及其PCR标记开发

doi:10.3864/j.issn.0578-1752.2019.01.001

中国农业科学 ›› 2019, Vol. 52 ›› Issue (1): 1-10.doi: 10.3864/j.issn.0578-1752.2019.01.001

• 作物遗传育种·种质资源·分子遗传学 • 下一篇

基于簇毛麦No.1026转录组的SSR序列分析及其PCR标记开发

陈竟男^1,²(),马晓兰¹,王振³,李仕金¹,谢皓²(),叶兴国¹,林志珊¹()

1 中国农业科学院作物科学研究所/农作物基因资源与基因改良国家重大科学工程/农业部麦类生物学与遗传育种重点实验室,北京 100081
2 北京农学院植物科学技术学院,北京 102206
3 北方民族大学生物工程与科学学院,银川 750021

收稿日期:2018-07-01 接受日期:2018-09-19 出版日期:2019-01-01 发布日期:2019-01-12
通讯作者: 谢皓,林志珊
基金资助:
国家重点研发计划(2016YFD0102002);国家重点研发计划(2016YFD0102001);中国农业科学院科技创新工程2060302-2-17年

SSR Sequences and Development of PCR Markers Based on Transcriptome of Dasypyrum villosum No.1026

CHEN JingNan^1,²(),MA XiaoLan¹,WANG Zhen³,LI ShiJin¹,XIE Hao²(),YE XingGuo¹,LIN ZhiShan¹()

1 Institute of Crop Science, Chinese Academy of Agricultural Sciences/National Key Facility of Crop Gene Resources and Genetic Improvement/Key Laboratory of Biology and Genetic Improvement of Triticeae Crops, Ministry of Agriculture, Beijing 100081
2 School of Plant Science and Technology, Beijing University of Agriculture, Beijing 102206
3 College of Bioscience & Bioengineering, North Minzu University, Yinchuan 750021;

Received:2018-07-01 Accepted:2018-09-19 Online:2019-01-01 Published:2019-01-12
Contact: Hao XIE,ZhiShan LIN

摘要/Abstract

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。【结果】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。

关键词: 簇毛麦, 转录组, EST-SSR, 异染色体系, 染色体定位, 遗传多样性

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 3rd, 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.

Key words: Dasypyrum villosum, transcriptome, EST-SSR, alien chromosome lines, chromosome localization, genetic diversity

陈竟男,马晓兰,王振,李仕金,谢皓,叶兴国,林志珊. 基于簇毛麦No.1026转录组的SSR序列分析及其PCR标记开发[J]. 中国农业科学, 2019, 52(1): 1-10.

CHEN JingNan,MA XiaoLan,WANG Zhen,LI ShiJin,XIE Hao,YE XingGuo,LIN ZhiShan. SSR Sequences and Development of PCR Markers Based on Transcriptome of Dasypyrum villosum No.1026[J]. Scientia Agricultura Sinica, 2019, 52(1): 1-10.

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重复单元大小 Repeat unit size	构成比例 Composition ratio (%)	重复基序（位点数） Repeat motif (locus number)	重复次数 Repeats
单核苷酸Mononucleotide	27.13	A/T(2124)、C/G(724)	10—23
双核苷酸Dinucleotide	18.39	AG/CT(1054)、AC/GT(546)、AT/AT(173)、CG/CG(157)	6—12
三核苷酸Trinucleotide	50.33	CCG/CGG(2201)、AGG/CCT(1003)、AGC/CTG(756)、 ACC/GGT(347)、AAG/CTT(322)、ACG/CGT(265)、ATC/ATG(159)、AAC/GTT(135)、AAT/ATT(48)、 ACT/AGT(47)	5—10
四核苷酸Tetranucleotide	3.75	AGGG/CCCT(46)、ACGC/GCGT (40)、ATCC/ATGG(37)、 AAGG/CCTT(35)、AGGC/GCCT(23)、ACAT/ATGT(21)、 AGCG/CGCT(21)、AAAG/CTTT(20)、AAAT/ATTT(17)、 AGCC/CTGG(16)、ACGG/CCGT(16)、CCCG/CGGG(15)、 AAAC/GTTT(14)、AGAT/ATCT(13)、AATC/ATTG(10)、 CCGG/CCGG(7)、AACC/GGTT(6)、ACAG/CTGT(5)、 AGCT/AGCT(4)、ATCG/ATCG(3)、ACTG/AGTC(3)、 ACTC/AGTG(3)、AATG/ATTC(3)、AAGC/CTTG(3)、 ATGC/ATGC(2)、ACGT/ACGT(2)、ACCG/CGGT(2)、 ACCC/GGGT(2)、AAGT/ACTT(2)、AACG/CGTT(2)、 ACCT/AGGT(1)	5—6
五核苷酸Pentanucleotide	0.34	AGAGG/CCTCT(5)、AGAGC/CTCTG(4)、AAGGG/CCCTT(2) 、ACAGC/CTGTG(2)、ACCAG/CTGGT(2)、AGGCG/CCTCG(2)、 ATCCC/ATGGG(2)、AAAAG/CTTTT(1)、AAACC/GGTTT(1)、 AAACT/AGTTT(1)、AAATC/ATTTG(1)、AAGCT/AGCTT(1)、 AAGGC/CCTTG(1)、AATAC/ATTGT(1)、ACACC/GGTGT(1)、ACCCC/GGGGT(1)、ACCGC/CGGTG(1)、ACGAG/CGTCT(1)、 ACGGC/CCGTG(1)、ACTAG/AGTCT(1)、AGCCC/CTGGG(1)、 AGCCG/CGGCT(1)、AGGGG/CCCCT(1)、ATCCG/ATCGG(1)	5—6
六核苷酸 Hexanucleotide	0.06	AAAGCC/GGCTTT(3)、AAACGC/CGTTTG(1)、AATGCC/ATTGGC(1)、ACCAGC/CTGGTG(1)、ACCAGC/CTGGTG(1)	6—8

基于簇毛麦No.1026转录组的SSR序列分析及其PCR标记开发

SSR Sequences and Development of PCR Markers Based on Transcriptome of Dasypyrum villosum No.1026

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