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

Abstract

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

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.

Figures/Tables 5

Table 1

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References 40

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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

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

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