基于转录组SNP构建油茶主要品种资源的分子身份证

doi:10.3864/j.issn.0578-1752.2023.02.002

摘要/Abstract

摘要：

【目的】近年来，油茶（Camellia oleifera）产业发展迅速，已成为中国四大油料之一。油茶良种不断涌现，但品质参差不齐，“同名异物、同物异名”等现象时有发生。建立油茶品种资源的单核苷酸多态性（single-nucleotide polymorphism，SNP）分子标记数据库，筛选重要SNP位点，开发油茶品种资源DNA指纹图谱，构建油茶品种资源的分子身份证，为品种鉴别、品种追溯等提供分子水平鉴别技术支撑。【方法】以221份普通油茶品种资源为材料，提取未成熟种子RNA，进行转录组测序。以二倍体南荣油茶基因组为参考，识别供试油茶品种资源的SNP位点并基因分型，利用SNP数据分析油茶群体及亚群的遗传多样性，分析SNP位点的观测杂合度、期望杂合度、多态信息含量（PIC）等信息，筛选核心SNP位点并采用Sanger测序验证，得到最优SNP位点组合后，结合品种资源基本信息构建油茶品种资源分子身份证。【结果】从油茶转录组中共检测到1 849 953个高质量SNP位点。群体遗传多样性分析发现，油茶群体观测杂合度为0.2966，期望杂合度为0.2462，固定指数为-0.2048，PIC为0.2073，最小等位基因频率为0.1648。参试群体的各亚群间遗传分化较小，存在较高的基因流，主要变异存在于亚群内。根据PIC、连锁不平衡衰退距离（LD）等参数从所有SNP位点中筛选出31个多态性高的核心位点，Sanger测序验证其中8个核心位点基因分型的准确率在91.36%以上。利用核心位点组成DNA指纹图谱，可区分出全部参试油茶品种资源。DNA指纹图谱结合油茶品种资源基本信息，构建成由66位数字组成的油茶品种资源分子身份证。【结论】依据SNP标记的PIC、LD等指标，筛选出31个核心SNP位点，精准区分全部供试油茶品种资源。将31个SNP位点所构建的油茶品种资源DNA指纹图谱与品种资源的起源、资源类型和亚群分布等基本属性信息相结合，构建了每份油茶品种资源唯一的分子身份证，并生成相应的条形码和二维码。

关键词: 普通油茶, 品种鉴别, 单核苷酸多态性, 指纹图谱, 分子身份证

Abstract:

【Objective】Camellia oleifera is a traditional woody oil plant and been widely cultivated in China. In order to facilitate the protection and precise management of C. oleifera cultivars and avoid the phenomenon of homonyms and synonyms, single-nucleotide polymorphism (SNP) marker database of C. oleifera cultivars was established, and a set of core SNPs were selected to construct molecular fingerprint and ID for each cultivar. 【Method】The RNA of developing seeds of 221 C. oleifera clones was extracted and RNA-seq were performed. Using C. oleifera var. ‘Nanyongensis’ genome sequence as reference, high-quality SNPs for C. oleifera were screened and the genotyping of accessions was carried out. Furthermore, the genetic diversity of C. oleifera population and subpopulations were analyzed using SNP data, including observed heterozygosity, expected heterozygosity and polymorphism information content (PIC) of the SNPs, etc. The SNP loci were further filtered by their polymorphism and location information to obtain the optimal combination of core SNP loci. Sanger-seq was performed to verify the core SNP loci. The fingerprints of each clone were formed according to the genotypes of the core SNPs. The molecular IDs of C. oleifera clones were finally constructed by combining the basic information and fingerprint of C. oleifera clones. 【Result】A total of 1 849 953 high-quality SNP loci were obtained from the transcriptomes of C. oleifera. The average values of observed heterozygosity, expected heterozygosity, fixed index, PIC and minor allele frequency of the C. oleifera population were 0.2966, 0.2462, -0.2048, 0.2073, and 0.1648, respectively. The genetic differentiation among the subpopulations of C. oleifera was minor with the high level gene flow, while the main variation was inside of the subpopulation. Filtered by PIC, LD, etc., 31 core SNP loci were screened out to distinguish all C. oleifera clones. The genotypes of all accessions in the eight core loci were further detected using Sanger-seq, and the verified rates were over 91.36%. All C. oleifera clones used in this study can be distinguished using the DNA fingerprints constructed by the 31 core SNPs. Based on the fingerprint of 31 SNP markers and the basic information of C. oleifera clones, a molecular ID of each clone, which composed of 66 digits, was formed finally. 【Conclusion】According to the polymorphism information of SNP markers, 31 core SNP loci were catched. And all C. oleifera clones were accurately distinguished. Furthermore, The DNA fingerprints of 221 C. oleifera clones were constructed by the 31 SNP markers. A unique molecular identity code for each germplasm was constructed using the DNA fingerprints and the converted serial codes from information of the C. oleifera clones. Finally, the bar codes and quick response (QR) codes are generated as the molecular ID, which can be quickly identified by the code scanning equipment.

Key words: Camellia oleifera, cultivar identification, single-nucleotide polymorphism (SNP), DNA fingerprint, molecular ID

林萍, 王开良, 姚小华, 任华东. 基于转录组SNP构建油茶主要品种资源的分子身份证[J]. 中国农业科学, 2023, 56(2): 217-235.

LIN Ping, WANG KaiLiang, YAO XiaoHua, REN HuaDong. Development of DNA Molecular ID in Camellia oleifera Germplasm Based on Transcriptome-Wide SNPs[J]. Scientia Agricultura Sinica, 2023, 56(2): 217-235.

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链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2023.02.002

https://www.chinaagrisci.com/CN/Y2023/V56/I2/217

图/表 11

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31个核心SNP位点及其详细信息"

序号 No.	核心位点 Loci	PIC	染色体 Chr.	等位基因 Alleles	位置 Location		所在的基因 Gene	基因注释 Gene annotation		功能类型 Function_type		功能 Function
1	SNP-1-16341109	0.4701	Chr.1	A/G	外显子 Exonic		Scaffold 1-snap-gene-163.21	异青霉素N合酶 Isopenicillin N synthase		非同义突变 Nonsynonymous		外显子1：c.A137G:p.N46S ^* Exon1: c.A137G:p.N46S
2	SNP-1-22551920	0.4890	Chr.1	C/T	5′非编码区 5′UTR		Scaffold 1-snap-gene-225.31	丝状植物蛋白7亚型X2 Filament-like plant protein 7 isoform X2		-		c.-1603G>A^#
3	SNP-1-34038354	0.4867	Chr.1	T/A	3′非编码区 3′UTR		Scaffold 1-snap-gene-340.25	GATA转录组因子28 GATA transcription factor 28-like		-		c.1337T>A^&
4	SNP-1-49052505	0.4941	Chr.1	A/G	外显子 Exonic		Scaffold 1-snap-gene-490.28	磷酸核糖胺-甘氨酸连接酶 Phosphoribosylamine-glycine ligase		同义突变 Synonymous		外显子5：c.T1368C:p.H456H Exon5:c.T1368C:p.H456H
5	SNP-1-110833857	0.4679	Chr.1	G/C	外显子 Exonic		Scaffold 1-snap-gene-1108.1	40S核糖体蛋白s24-1 40S ribosomal protein s24-1		同义突变 Synonymous		外显子3：c.C117G:p.A39A Exon3:c.C117G:p.A39A
6	SNP-1-115890029	0.4882	Chr.1	A/G	外显子 Exonic		Scaffold 1-processed-gene-1159.31	-		同义突变 Synonymous		外显子1：c.T369C:p.G123G Exon1:c.T369C:p.G123G
7	SNP-1-188555309	0.4968	Chr.1	T/G	3′非编码区 3′UTR		Scaffold 1-snap-gene-1885.48	ATP依赖的DNA解旋酶2亚基KU80 ATP-dependent DNA helicase 2 subunit KU80		-		c. 104A>C
8	SNP-2-4697104	0.4656	Chr.2	T/A	5′非编码区 5′UTR		Scaffold 2-snap-gene-47.0	通用应激蛋白a相似蛋白 Universal stress protein a-like protein		-		c.-51T>A
9	SNP-2-102046792	0.4620	Chr.2	C/A	3′非编码区 3′UTR		Scaffold 2-snap-gene-1020.8	胼胝质合成酶12 Callose synthase 12		-		c.121C>A
10	SNP-3-58564519	0.4883	Chr.3	C/T	外显子 Exonic		Scaffold 3-processed-gene-585.38	未知蛋白LOC103487192 Uncharacterized protein LOC103487192		同义突变 Synonymous		外显子3：c.G201A:p.V67V Exon3:c.G201A:p.V67V
11	SNP-4-5589980	0.4644	Chr.4	T/G	外显子 Exonic		Scaffold 4-snap-gene-56.9	嘌呤通透酶4 Probable purine permease 4		同义突变 Synonymous		外显子2：c.A1131C:p.G377G Exon2:c.A1131C:p.G377G
12	SNP-5-59238652	0.4836	Chr.5	G/C	外显子 Exonic		Scaffold 5-snap-gene-592.4	40s核糖体蛋白s8 40s ribosomal protein s8		同义突变 Synonymous		外显子3：c.C417G:p.V139V Exon3:c.C417G:p.V139V
13	SNP-5-135461353	0.4702	Chr.5	T/C	外显子 Exonic		Scaffold 5-snap-gene-1354.5	驱动蛋白近似蛋白KIN-7N Kinesin-like protein KIN-7N		同义突变 Synonymous		外显子17：c.A1842G:p.S614S Exon17:c.A1842G:p.S614S
14	SNP-5-175443498	0.4721	Chr.5	T/G	5′非编码区 5′UTR		Scaffold 5-processed-gene-1754.17	黄酮3-羟化酶 Flavanone 3-hydroxylase		-		c.-19T>G
15	SNP-6-123202382	0.4872	Chr.6	A/C	外显子 Exonic		Scaffold 6-snap-gene-1232.1	RNA聚合酶Ⅱ转录亚基1中间体 Mediator of RNA polymerase II transcription subunit 1		非同义突变 Nonsynonymous		外显子8：c.A1694C:p.K565T Eon8:c.A1694C:p.K565T
16	SNP-6-125991240	0.4871	Chr.6	T/C	外显子 Exonic		Scaffold 6-snap-gene-1260.11	寡肽转运体4 Oligopeptide transporter 4		非同义突变 Nonsynonymous		外显子3：c.T821C:p.V274A Exon3:c.T821C:p.V274A
17	SNP-7-134929316	0.4606	Chr.7	T/C	外显子 Exonic		Scaffold 7-snap-gene-1349.44	辅素样蛋白1亚型 Auxilin-like protein 1 isoform X1		同义突变 Synonymous		外显子2：c.A2022G:p.Q674Q Exon2:c.A2022G:p.Q674Q
序号 No.	核心位点 Loci	PIC	染色体 Chr.	等位基因 Alleles	位置 Location	所在的基因 Gene		基因注释 Gene annotation		功能类型 Function_type		功能 Function
18	SNP-8-37227126	0.4672	Chr.8	C/T	外显子 Exonic		Scaffold 8-snap-gene-372.3	2-甲基丁醛肟单氧化酶 2-methylbutanal oxime monooxygenase		非同义突变 Nonsynonymous		外显子2：c.G949A:p.V317I Exon2:c.G949A:p.V317I
19	SNP-9-10583849	0.4926	Chr.9	A/T	外显子 Exonic	Scaffold 9-snap-gene-105.30		磷脂转运ATP酶4 Probable phospholipid-transporting ATPase 4	同义突变 Synonymous		外显子:7：c.T2460A:p.T820T Exon7:c.T2460A:p.T820T
20	SNP-9-23224047	0.4819	Chr.9	T/C	外显子 Exonic	Scaffold 9-snap-gene-232.15		水解酶6结构域包含蛋白 Abhydrolase_6 domain-containing protein	同义突变 Synonymous		外显子3：c.T450C:p.L150L Exon3:c.T450C:p.L150L
21	SNP-9-89305797	0.4999	Chr.9	T/C	外显子 Exonic	Scaffold 9-snap-gene-893.6		抗病性蛋白 Disease resistance protein	非同义突变 Nonsynonymous		外显子2：c.A496G:p.S166G Exon2:c.A496G:p.S166G
22	SNP-9-95564863	0.4817	Chr.9	G/T	外显子 Exonic	Scaffold 9-snap-gene-955.13		复制蛋白A 70kDa DNA结合亚基B Replication protein A 70 kDa DNA-binding subunit B	非同义突变 Nonsynonymous		外显子13：c.C1768A:p.H590N Exon13:c.C1768A:p.H590N
23	SNP-10-146191194	0.4586	Chr.10	G/C	外显子 Exonic	Scaffold 10-processed-gene-1462.14		未知蛋白LOC100853376 Uncharacterized protein LOC100853376	非同义突变 Nonsynonymous		外显子1：c.G85C:p.D29H Exon1:c.G85C:p.D29H
24	SNP-11-126349400	0.4999	Chr.11	C/T	外显子 Exonic	Scaffold 11-snap-gene-1263.11		未知蛋白LOC107417301亚型X1 Uncharacterized protein LOC107417301 isoform X1	同义突变 Synonymous		外显子3：c.C1398T:p.F466F Exon3:c.C1398T:p.F466F
25	SNP-11-150316502	0.4675	Chr.11	G/A	外显子 Exonic	Scaffold 11-snap-gene-1503.37		细胞色素P450 CYP72A219 Cytochrome P450 CYP72A219	同义突变 Synonymous		外显子6：c.G465A:p.Q155Q Exon6:c.G465A:p.Q155Q
26	SNP-11-175529761	0.4925	Chr.11	G/C	外显子 Exonic	Scaffold 11-snap-gene-1755.2		mRNA去壳蛋白4增强子 Enhancer of mRNA-decapping protein 4-like	非同义突变 Nonsynonymous		外显子12：c.G4011C:p.K1337N Exon12:c.G4011C:p.K1337N
27	SNP-12-38432972	0.4699	Chr.12	C/G	外显子 Exonic	Scaffold 12-snap-gene-384.1		多药物和有毒物质排出转运 Multidrug and toxic extrusion transporter	同义突变 Synonymous		外显子1：c.G132C:p.S44S Exon1:c.G132C:p.S44S
28	SNP-12-88588135	0.4907	Chr.12	G/A	5′非编码区 5′UTR	Scaffold 12-snap-gene-885.8		网状内皮素相似蛋白B5 Reticulon-like protein B5	-		c.-74C>T
29	SNP-13-180557942	0.4952	Chr.13	T/C	外显子 Exonic	Scaffold 13-snap-gene-1805.53		富含半胱氨酸/组氨酸的C1结构域家族蛋白 Cysteine/Histidine-rich C1 domain family protein	同义突变 Synonymous		外显子3：c.T1047C:p.Y349Y Exon3:c.T1047C:p.Y349Y
30	SNP-14-86914708	0.4819	Chr.14	T/C	外显子 Exonic	Scaffold 14-snap-gene-869.1		纤溶酶原激活蛋白抑制因子1 RNA结合蛋白 Plasminogen activator inhibitor 1 RNA-binding protein	同义突变 Synonymous		外显子4：c.T672C:p.D224D Exon4:c.T672C:p.D224D
31	SNP-14-87149383	0.4983	Chr.14	G/T	外显子 Exonic	Scaffold 14-snap-gene-871.12		蛋白霜霉病抗性6 Protein downy mildew resistance 6	非同义突变 Nonsynonymous		外显子3：c.G659T:p.S220I Exon3:c.G659T:p.S220I

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群体 Pop	样本数 Sample No.	Na	Ne	Ho	He	F-index	Nei	π	PIC	Shannon	MAF	SNP位点数 SNP No.
亚群Ⅰ Subpop Ⅰ	46	1.3064	1.3728	0.2958	0.2431	-0.2169	0.2464	0.000158	0.2039	0.3922	0.1654	1849953
亚群Ⅱ Subpop Ⅱ	31	1.3064	1.374	0.2973	0.2427	-0.2250	0.2475	0.000158	0.2032	0.3903	0.1679	1849953
亚群Ⅲ Subpop Ⅲ	38	1.3006	1.3662	0.2906	0.2383	-0.2198	0.2422	0.000155	0.1998	0.3845	0.1638	1849953
亚群Ⅳ Subpop Ⅳ	35	1.307	1.3717	0.2965	0.2417	-0.2268	0.246	0.000157	0.2025	0.3893	0.1664	1849953
亚群Ⅴ Subpop Ⅴ	42	1.3104	1.3772	0.3011	0.2449	-0.2297	0.2484	0.000159	0.205	0.3939	0.1678	1849953
亚群Ⅵ Subpop Ⅵ	15	1.3105	1.3751	0.3013	0.2396	-0.2578	0.2497	0.000160	0.1992	0.3807	0.1817	1849925
亚群Ⅶ Subpop Ⅶ	8	1.2989	1.3573	0.2893	0.2234	-0.2950	0.2426	0.000155	0.1840	0.3484	0.2076	1843321
亚群Ⅷ Subpop Ⅷ	5	1.3037	1.3504	0.2945	0.2102	-0.4010	0.2397	0.000152	0.1700	0.3186	0.2526	1774982
全体 All	220	1.3062	1.3739	0.2966	0.2462	-0.2048	0.2468	-	0.2073	0.3991	0.1648	1849953

群体 Pop	亚群Ⅰ Subpop Ⅰ	亚群Ⅱ Subpop Ⅱ	亚群Ⅲ Subpop Ⅲ	亚群Ⅳ Subpop Ⅳ	亚群Ⅴ Subpop Ⅴ	亚群Ⅵ Subpop Ⅵ	亚群Ⅶ Subpop Ⅶ	亚群Ⅷ Subpop Ⅷ
亚群Ⅰ SubpopⅠ	-	0.0015	0.0040	0.0022	0.0047	0.0000	0.0000	0.0058
亚群Ⅱ Subpop Ⅱ	161.8740	-	0.0035	0.0010	0.0028	0.0001	0.0000	0.0064
亚群Ⅲ Subpop Ⅲ	62.6660	71.1826	-	0.0034	0.0046	0.0015	0.0000	0.0085
亚群Ⅳ Subpop Ⅳ	115.9860	247.3711	73.8063	-	0.0033	0.0000	0.0000	0.0060
亚群Ⅴ Subpop Ⅴ	52.9548	88.4173	53.6558	74.4734	-	0.0004	0.0000	0.0080
亚群Ⅵ Subpop Ⅵ	-	4343.6263	168.3339	-	686.0412	-	0.0000	0.0045
亚群Ⅶ Subpop Ⅶ	-	-	-	-	-	-	-	0.0012
亚群Ⅷ Subpop Ⅷ	42.7958	38.8365	29.1307	41.2443	30.9227	55.1133	217.1238	-

位点 Loci	正向引物 Forward Primer (5'-3')	反向引物 Reverse Primer (5'-3')	退火温度 Tm (℃)	产物 Product (bp)	SNP在产物上的位置 SNP location on the product (bp)
SNP-1-34038354	CGTGTCACAACCTAGTTCCC	ACCAAGGACGCAGTTCTTC	40.7	350	216
SNP-1-110833857	AGAGATTAAGATGTAACAACCAGT	CACTGTCCTCAGATAATTGATGTT	36.7	425	274
SNP-11-175529761	AGGGGTAGTGTTTCTTGGATTTTC	CAGGTCATCAGCATGGAGTTG	44.3	404	198
SNP-14-86914708	GGAATGAATTTAAACGTGATGGT	TTCTCTACTTCTCACTACATACTT	38	434	273
SNP-14-87149383	TTGGCTCCGACATACCTAAGA	GCAGGCTCAACCACTTCAT	47.8	383	182
SNP-2-4697104	GGACACCAGACAGAGGAATAATC	GTTATCCACAGCCCACTTCAG	49.7	372	241
SNP-5-175443498	ATCACACTGTAGTAGCGGACAA	CACACGCCTCCACAATCTTC	46.8	455	241
SNP-9-23224047	ACGGTGATGCTGTGTTCTT	TGCTTGGAATAATTGAATCCTGTG	38.2	437	177