Scientia Agricultura Sinica ›› 2022, Vol. 55 ›› Issue (15): 2875-2882.doi: 10.3864/j.issn.0578-1752.2022.15.001

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

PSORA: A Strategy Based on High-Throughput Sequence for Analysis of T-DNA Insertion Sites

MA XueMeng(),YU ChengMin(),SAI XiaoLing,LIU Zhen,SANG HaiYang,CUI BaiMing()   

  1. College of Life Sciences, Shihezi University, Shihezi 832003, Xinjiang
  • Received:2022-03-19 Accepted:2022-05-10 Online:2022-08-01 Published:2022-08-02
  • Contact: BaiMing CUI E-mail:937696167@qq.com;2373151062@qq.com;cbmzyy@sina.com

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Abstract:

【Objective】The purpose of this study was to establish a simple and efficient approach for identifying all T-DNA insertion sites. 【Method】A T-DNA insertion sites analysis approach based on high-throughput sequence technologies was developed, called PSORA: Parallel sequencing of one round amplicons. The process involves high-throughput amplicon sequencing of a round of thermal asymmetric PCR (TAIL-PCR) and bioinformatics analysis of T-DNA insertion sites, which reduces concerns about the specificity of TAIL-PCR. In PSORA, only two primers are required, a degenerate primer and a T-DNA specific primer. A 6-nt Barcode was designed at the 5’ end of the specific primers for labeling different transgenic events. All five transgenic events (L1, L6, L9, L15 and L19) of tobacco used in this study were produced via Agrobacterium mediated transformation with plasmids pBI121. In addition, the results of PSORA are confirmed by standard PCR. 【Result】The T-DNA insertion sites of five transgenic events were analyzed by PSORA. The results showed that L6 contained two insertion sites (36 316 bp on NW_015801367 and 42 202 bp on NW_015950898), the lines of L9, L15 and L19 each contained one insertion site (The insertion site of L9 was located at 235 969 bp on NW_015943682. The insertion site of L15 was located at 60 529 bp on NW_015802951 and the insertion site of L19 was located at 12 188 bp on NW_015863435), but the insertion site of L1 could not be detected. PCR was performed to validate the results from bioinformatics analysis, transgenic events with different insertion sites were used as negative controls for each other, and the wild type (WT) was used as a blank control. The results showed that specific amplification consistent with expectations was obtained in each transgenic event. The effectiveness of PSORA was successfully confirmed. 【Conclusion】PSORA is an effective strategy to analyze T-DNA insertion sites. PSORA can parse the comprehensive molecular characteristics of all T-DNA insertion events simultaneously, making it simpler and faster than the traditional methods of genome walking.

Key words: genome walking, T-DNA insertion sites, transgenic plant, high-throughput sequencing, TAIL-PCR

Table 1

The sequence of primers"

类型
Type 		引物
Primer		 引物序列
Sequence of primers (5′-3′)		 对应的样品
Corresponding sample
简并引物
Degenerated primer		 AP1 GARMSNCCNAG L1、L6、L9、L15、L19
AP2 YTCSKNGANGC L1、L6、L9、L15、L19
特异性引物
Specific primer		 BSP-1 TCTCACCAGATTGTCGTTTCCCGCCT L1
BSP-2 TCACGCCAGATTGTCGTTTCCCGCCT L6
BSP-3 CGTCTACAGATTGTCGTTTCCCGCCT L9
BSP-4 CGCACCCAGATTGTCGTTTCCCGCCT L15
BSP-5 TAGATCCAGATTGTCGTTTCCCGCCT L19
SHP-1 GGAAACGACAATCTGACTCACCAGATTGTCGTTTCCCGCCT L1
SHP-2 GGAAACGACAATCTGTCACGCCAGATTGTCGTTTCCCGCCT L6
SHP-3 GGAAACGACAATCTGCGTCTACAGATTGTCGTTTCCCGCCT L9
SHP-4 GGAAACGACAATCTGCTCACCCAGATTGTCGTTTCCCGCCT L15
SHP-5 GGAAACGACAATCTGGAGATCCAGATTGTCGTTTCCCGCCT L19
检测引物
Detection primer		 TRSP_F TTATGGAACGTCAGTGGAGCAT 通用 Universal
NW_015801367_R CCAATGGGAATGGGGCAAATG L6
NW_015950898_R AATGTGGTTGCTTGCCGTTG L6
NW_015943682_R ACTAGATGGGATGCCCAAGATT L9
NW_015802951_R TTCATAACATGAAACACCATAATAG L15
NW_015863435_R ACAGAAACAGGACGGAAGTGA L19

Table 2

Cycling conditions for PCR"

阶段Stage 循环条件Cycle conditions 循环次数Cycle times
预变性Pre-denaturation 94℃ 3 min 1
第一阶段
First stage		 94℃ 30 s;58℃ 30 s;72℃ 3 min 4
94℃ 30 s;80℃ 5 min;25℃（1℃ s-1）3 min;72℃ 3 min 1
第二阶段
Second stage		 94℃ 5 s;68℃ 3 min;94℃ 5 s;68℃ 3 min;
94℃ 30 s;40℃（1℃ s-1）1 min;72℃ 3 min		 25
延伸Extension 72℃ 10 min 1

Fig. 1

Schematic of primer annealing Specific primers anneal to the T-DNA region, degenerate primers are expected to anneal to flanking unknown sequences. A: BSP and AP anneal to template; B: SHP anneal to template in high-stringency cycles; C: SHP forms hairpin structure in low-stringency cycle/ reduced-stringency cycles"

Table 3

The comparison of data from two libraries"

文库Libraries GWHP GWBP
特异性引物的序列差异
Sequence differences of specific primers		 HSP的5′端具有特异性反向互补序列
There is a specific reverse complementary sequence in the 5′ end of HSP		 BSP不含特异性反向互补序列
There is no specific reverse complementary sequence in BSP
总reads数量（对） Number of total reads (pair) 24 673 038 19 000 877
特异性引物产生的非特异性扩增产物数量（对）
Number of nonspecific amplification products produced by specific primers (pair)		 9 628 240 12 862 627
特异性引物产生的非特异性扩增产物在总reads中的比例
Proportion of nonspecific amplification products produced by specific primers in total reads (%)		 39.02 67.69
含特异性序列总reads数量（对）
Total reads with specific sequences (pair)		 9 631 972 12 865 999
特异性扩增reads数量（对）
Number of reads produced by specific amplification (pair)		 3 732 3 372
特异性扩增reads在含特异性序列总reads中的比例
Proportion of reads produced by specific amplification in total reads containing specific sequences (%)		 0.039 0.026

Fig. 2

The sequences related with insertion site Sequences near the right border of T-DNA are shown in red, tobacco genome sequences are shown in green, consensus sequences are shown in black letters, and intermediate junction sequences are shown in grey"

Fig. 3

The electrophoresis results of insertion site by PCR"

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