小麦Glu-3位点基因拷贝数的变异分析

doi:10.3864/j.issn.0578-1752.2021.06.002

摘要/Abstract

摘要：

【目的】基因拷贝数变异是一种常见又重要的基因结构变异,往往影响个体表型。低分子量麦谷蛋白（low-molecular-weight glutenin subunit,LMW-GS）是小麦贮藏蛋白的主要组成部分,位于Glu-3位点。小麦作为异源六倍体,其庞大且复杂的基因组结构导致难以利用传统方法检测目的基因的拷贝数,针对小麦基因组,筛选可靠稳定的内参基因和体系,探索适合复杂基因组的拷贝数变异测定技术,测定Glu-3位点LWM-GS基因拷贝数。【方法】以Acc1为内参基因,根据基因序列设计内参引物和探针,通过定性和定量PCR测定内参基因在12个普通小麦品种中的拷贝数,分析该基因拷贝数在不同品种间的稳定性;又以小麦品种篙优2018的5个稀释浓度的基因组DNA为模板,利用qRT-PCR验证Acc1内参系统的重复性和准确性;根据Glu-A3位点LMW-GS基因序列设计特异性引物及探针,利用qRT-PCR和ddPCR 2种方法检测8个小麦品种Glu-A3位点基因拷贝数,比较后选择更优的高通量基因拷贝数检测方法;再根据Glu-B3和Glu-D3位点LMW-GS基因序列设计相应的特异性引物及探针,并利用ddPCR技术检测和分析了231份小麦品种的Glu-A3、Glu-B3和Glu-D3位点上LMW-GS基因拷贝数。【结果】Acc1在12个普通小麦品种间、同一品种5个DNA稀释浓度间的拷贝数测定结果一致,技术重复间的变异系数仅为0.07%—0.77%,所构建的Acc1内参系统稳定;比较qRT-PCR和ddPCR 2种拷贝数检测方法,8个品种所测的Glu-A3位点拷贝数结果一致,分别为3、5、3、4、3、3、3和3;且ddPCR检测重复间的变异系数为0.30%—1.67%,远低于qRT-PCR的3.14%—12.72%,更加可靠;利用ddPCR对231份普通小麦品种的Glu-A3、Glu-B3和Glu-D3位点上LMW-GS基因拷贝检测后分析发现,大多数小麦品种在3个位点上的拷贝数为4,所占频率分别为51.95%、32.03%和28.57%,Glu-3位点总拷贝数变异范围为10—21,变异系数为16.12%。【结论】Acc1内参系统具有良好的稳定性和重复性,可以用作小麦Glu-3位点和其他目的基因拷贝数检测的内参;qRT-PCR和ddPCR均可用于小麦基因拷贝数的检测,但后者更稳定、可靠,且操作简单、检测通量高。

关键词: 小麦, 低分子量麦谷蛋白, 微滴式数字PCR, 拷贝数变异

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)

陈璨,韩南南,刘洋,史晓维,司红起,马传喜. 小麦Glu-3位点基因拷贝数的变异分析[J]. 中国农业科学, 2021, 54(6): 1092-1103.

Can CHEN,NanNan HAN,Yang LIU,XiaoWei SHI,HongQi SI,ChuanXi MA. Analysis of Copy Number Variation of Glu-3 Locus in Common Wheat[J]. Scientia Agricultura Sinica, 2021, 54(6): 1092-1103.

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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

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 (%)
DNA浓度 DNA concentration (ng·μL^-1)	1	2	3	平均值 Average value	标准差 Standard deviation	方差 Variance	变异系数 Coefficient of variation (%)
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

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
品种名 Sample	Ct (Glu-A3)	Ct (Acc1)	3X₀/R₀	Ct (Glu-A3)	Ct (Acc1)	3X₀/R₀	Ct (Glu-A3)	Ct (Acc1)	3X₀/R₀	变异系数 Coefficient of variation	拷贝数 Copy number
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