Scientia Agricultura Sinica ›› 2021, Vol. 54 ›› Issue (6): 1092-1103.doi: 10.3864/j.issn.0578-1752.2021.06.002

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

Analysis of Copy Number Variation of Glu-3 Locus in Common Wheat

Can CHEN(),NanNan HAN(),Yang LIU,XiaoWei SHI,HongQi SI(),ChuanXi MA()   

  1. College of Agronomy, Anhui Agriculture University/Key Laboratory of Wheat Biology and Genetic Improvement on Southern Yellow & Huai River Valley, Ministry of Agriculture and Rural Affairs, Hefei 230036
  • Received:2020-07-28 Accepted:2020-09-27 Online:2021-03-16 Published:2021-03-25
  • Contact: HongQi SI,ChuanXi MA E-mail:chencan-L@163.com;1565635050@qq.com;sihq2002@163.com;machuanxi@ahau.edu.cn

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Abstract:

【Objective】The variations in numbers of copies of a gene is a common and important gene structure variation, which often effects individual phenotype. Low-molecular-weight glutenin subunit (LMW-GS) located at the Glu-3 loci, is an essential part of storage protein in wheat. As a hetero-hexaploid, the huge and complex wheat genome makes it difficult to detect numbers of gene copies by classical methods. A limited information is available about CNV analysis of LWM-GS. To screen reliable and stable internal reference genes and systems for complex genome and explore the CNV determination technology suitable in different wheat varieties, the techniques used for detection of gene copy numbers, based on droplet digital PCR (ddPCR) which improve the detection flux of target gene and determine the particular numbers of gene copies of Glu-3 loci. 【Method】In this paper, Acc1 was used as the internal reference gene. The internal reference primers and probes were designed by using the corresponding gene sequence. In order to analyze stability of numbers of copies of Acc1 in 12 common wheat varieties, we used qualitative and quantitative PCR. The genomic DNA of variety Gaoyou2018 with five different dilutions was used as template, and qRT-PCR was used to analyze the repeatability and accuracy of Acc1 internal reference system (primers and probes).The corresponding specific primers and probes were designed according to the LMW-GS gene sequence of Glu-A3 loci. Two different methods, qRT-PCR and ddPCR, were used to detect the numbers of copies of Glu-A3 loci in eight wheat varieties, in order to determine which method is more suitable for high-throughput detection of Glu-3 loci gene. The specific primers and probes were also designed according to the sequences of LMW-GS gene at Glu-B3 and Glu-D3 loci. The numbers of copies of LMW-GS gene at Glu-A3, Glu-B3 and Glu-D3 loci of 231 varieties were determined and analyzed by ddPCR. 【Result】The results showed that the gene copy numbers of Acc1 was consistent among varieties and different DNA concentrations of the same variety. The coefficient of variation (CV) between repeats was 0.07%-0.77%. It is also indicated from results that the Acc1 internal reference system constructed in this paper has good stability and repeatability. The results of qRT-PCR and ddPCR were consistent in detecting the numbers of copies of LMW-GS gene at Glu-A3 loci in 8 wheat varieties, which were 3, 5, 3, 4, 3, 3, 3 and 3, respectively. However, the CV among repeats detected by ddPCR was 0.30%-1.67%, which much lower than that by qRT-PCR. It showed that using ddPCR method to detect the gene copy numbers of Glu-3 loci gene is more stable and reliable. The numbers of copies of LMW-GS gene were 4 at Glu-A3, Glu-B3 and Glu-D3 having a frequency of 95%, 32.03% 28.57% respectively in 231 wheat varieties. The total variation range in numbers of copies of Glu-3 was 10-21, and the CV was 16.12%.【Conclusion】With good stability and repeatability, Acc1 could be used as internal reference gene for numbers of gene copies detection in wheat. Both qRT-PCR and ddPCR could be used in gene copy numbers detection with wheat genes, but the ddPCR is more simple, stable, reliable and has high detection flux.

Key words: wheat, low-molecular-weight glutenin subunit (LMW-GS), droplet digital PCR (ddPCR), copy number variation (CNV)

Table 1

The specific primer and probe sequence"

引物/探针名称
Primer/probe name		 序列
Sequence (5′-3′)		 退火温度
Annealing temperature (℃)
Glu-A3F AGCAATCCCGCCATGAGTCA
60
Glu-A3R AGTGGTGGTTTCGTACAACG
Glu-A3T AATGTGTCTCCCAACCCCAACAGCAG
Glu-B3F CAAGTCATCTTTAGCAAGCATCAGG
59
Glu-B3R AAGGTCTTCATGGTGGACTAGTGTT
Glu-B3T ATAGTAGCCAGGGCACCACCTCTTT
Glu-D3F ACTAATCGAGCATATCCTAA
51
Glu-D3R GTTACATTGGGTTAGGTTT
Glu-D3T AGCCTATACAAGGTTCCAAACTCGG
Acc1F GCAATGTAGCTGCGCTTCAC
Acc1R CCTGCAACCGTGGATTAAGT
T-Acc1 ACAGTAGCAGCACCAACATAACCCACAGC

Fig. 1

Agarose gel electrophoresis of the testing reference gene copy number stability 1: Qian 11240-2; 2: Zhengmai 583; 3: Zhongmai 14; 4: Dehongfumai 2; 5: Ruiquanmai 168; 6: Zhonghe-75; 7: Yikemai 5; 8: Neimai 836; 9: Yangguang838; 10: Annong 1020; 11: Xinmai 31; 12: Qian 090304-4"

Fig. 2

Testing reference gene copy number stability with qRT-PCR A: Reference gene qRT-PCR amplification curve of 12 different varieties; 1: Qian 11240-2; 2: Zhengmai 583; 3: Zhongmai 14; 4: Dehongfumai 2; 5: Ruiquanmai 168; 6: Zhonghe-75; 7: Yikemai 5; 8: Neimai 836; 9: Yangguang838; 10: Annong 1020; 11: Xinmai 31; 12: Qian 090304-4. B: Ct values obtained from 12 different varieties of common wheat"

Table 2

Reproducibility of Ct measurements using RT-PCR for five levels of concentration of genomic DNA from common wheat"

DNA浓度
DNA concentration (ng·μL-1)		 Ct值 Ct value 平均值
Average value		 标准差
Standard deviation		 方差
Variance		 变异系数
Coefficient of variation (%)
1 2 3
300 27.16 27.55 27.22 27.31 0.2100 0.0441 0.77
200 27.96 28.08 28.10 28.05 0.0757 0.0057 0.27
100 30.20 30.21 30.17 30.19 0.0208 0.0004 0.07
50 30.84 30.96 30.91 30.90 0.0603 0.0036 0.20
30 31.02 31.09 30.81 30.97 0.1457 0.0212 0.47

Fig. 3

Agarose gel of amplification products obtained with primer A: Detected with Glu-A3F/R primers. 1: Chinese Spring; 2: T. monococcum; 3: Ae.speltoides Tausch; 4: Aegilops tauschii Coss.; 5: T.urartu; 6: ddH2O; 7: Annong0711. B: Detected with Glu-B3F/R primers. 1: T. monococcum; 2: Aegilops tauschii Coss.; 3: T. turgidum; 4: Chinese Spring; 5: ddH2O. C: Detected with Glu-D3F/R primers. 1: T.urartu; 2: Ae.speltoides Tausch; 3: Aegilops tauschii Coss.; 4: T. turgidum; 5: ddH2O"

Fig. 4

Standard curves of the target gene and reference gene"

Table 3

Ct values and target gene copy numbers obtained from 8 varieties samples by three times repeat"

品种名
Sample		 重复1 Repeat 1 重复2 Repeat 2 重复3 Repeat 3 变异系数
Coefficient of variation		 拷贝数
Copy number
Ct
(Glu-A3)		 Ct
(Acc1)		 3X0/R0 Ct
(Glu-A3)		 Ct
(Acc1)		 3X0/R0 Ct
(Glu-A3)		 Ct
(Acc1)		 3X0/R0
烟农15
Yangnong 15		 27.95 28.55 2.52 28.21 28.97 2.86 27.89 28.63 2.82 6.80 3
安农92484W
Annong 92484W		 26.58 27.87 4.50 26.71 27.93 4.24 26.63 27.91 4.45 3.14 5
百农64
Bainong 64		 27.45 28.33 3.18 27.06 27.96 3.25 27.37 28.18 3.00 4.10 3
小偃6号
Xiaoyan 6		 27.41 28.41 3.51 27.11 28.09 3.47 27.30 28.14 3.09 6.91 4
周麦16
Zhoumai 16		 27.01 27.57 2.35 26.64 27.35 2.80 26.59 27.25 2.69 8.98 3
02P67 26.81 27.46 2.66 26.74 27.22 2.32 26.46 27.01 2.46 6.90 3
邯郸6172
Handan 6172		 27.50 27.92 2.19 27.58 28.32 2.83 27.52 28.12 2.53 12.72 3
周麦18
Zhoumai 18		 27.84 28.60 2.87 27.62 28.36 2.83 27.57 28.18 2.55 6.34 3

Fig. 5

Target gene copy numbers obtained from 8 varieties samples by ddPCR 1: Yannong15; 2: Annong 92848W; 3: Bainong64; 4: Xiaoyan6; 5: Zhoumai16; 6: 02P67; 7: Handan6172; 8: Zhoumai18"

Fig. 6

Glu-A3, Glu-B3 and Glu-D3 gene copy numbers frequency distribution of 231 varieties common wheat"

Fig. 7

Glu-3 gene copy numbers frequency distribution of 231 varieties common wheat"

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