利用荧光标记SSR鉴别21个茶花新品种

doi:10.11983/CBB18019

植物学报 ›› 2019, Vol. 54 ›› Issue (1): 37-45.DOI: 10.11983/CBB18019 cstr: 32102.14.CBB18019

利用荧光标记SSR鉴别21个茶花新品种

陶乃奇¹,张斌^2,³,刘信凯⁴,周和达²,钟乃盛⁴,严丹峰⁴,张敏¹,高继银⁴,张文驹^1,^*()

1 复旦大学生命科学学院生物多样性科学研究所, 生物多样性与生态工程教育部重点实验室, 上海 200433
2 上海星源农业实验场, 上海 201417
3 上海市农业发展促进中心, 上海 200335
4 棕榈生态城镇发展股份有限公司, 广州 510627

收稿日期:2018-01-18 接受日期:2018-04-28 出版日期:2019-01-01 发布日期:2019-07-31
通讯作者: 张文驹
基金资助:
国家自然科学基金(31670223);上海市农委农业种质资源创新与良种良法配套技术集成应用沪农科种字(2015第10号No.2016F2FC0012)

Identification of 21 New Camellia Hybrid Varieties by Fluorescence-labelled Simple Sequence Repeat Markers

Naiqi Tao¹,Bin Zhang^2,³,Xinkai Liu⁴,Heda Zhou²,Naisheng Zhong⁴,Danfeng Yan⁴,Min Zhang¹,Jiyin Gao⁴,Wenju Zhang^1,^*()

1 Ministry of Education Key Laboratory for Biodiversity Science and Ecological Engineering, Institute of Biodiversity Science, School of Life Sciences, Fudan University, Shanghai 200433, China
2 Shanghai Xingyuan Agricultural Experiment Center, Shanghai 201417, China
3 Shanghai Agricultural Development and Promotion Center, Shanghai 200335, China
4 Palm Eco-Town Development Co, Ltd, Guangzhou 510627, China

Received:2018-01-18 Accepted:2018-04-28 Online:2019-01-01 Published:2019-07-31
Contact: Wenju Zhang

摘要/Abstract

摘要： 利用杜鹃红山茶(Camellia azalea)转录组数据和前人研究中获得的12个高多态性的SSR位点, 采用荧光毛细管电泳分析了21个茶花杂交新品种的基因型。结果显示, 12对SSR引物都能获得稳定清晰的扩增产物, 且上述12个SSR位点的基因型能够完全区分上述21个茶花新品种。其中, 来自同一杂交组合后代的多个品种, 基因型间有差异的位点数为2-10个, 来自不同杂交组合后代的品种间, 其基因型有差异的位点数为5-12个。研究结果表明, 上述21个茶花新品种均获得了独特的基因型标记, 能准确地进行品种身份鉴定, 这对以扦插或嫁接扩大生产的茶花品种的鉴定和品种权保护具有重要意义。

关键词: 山茶属, 荧光标记SSR, 新品种保护, 品种鉴定

Abstract: In this study, 12 highly polymorphic simple sequence repeat (SSR) loci were selected from Camellia azalea transcriptome data and previous studies, and the genotypes of 21 new Camellia hybrid varieties were analyzed by fluorescence capillary electrophoresis. Repeatable and distinct amplification products could be obtained at 12 SSR loci from all samples, and 21 new varieties could be identified by the specific genotype of 12 SSR loci. Significantly, for the 21 new varieties, the number of discrepant loci was 2 to 10 among varieties from the same parental combination but 5 to 12 among varieties from different combinations. Hence, each of the 21 varieties was marked by a unique group of molecular markers, which can be used to identify these varieties accurately. These markers are very useful for identifying and protecting Camellia varieties, especially for those propagated that are produced by grafting or cutting.

Key words: Camellia, fluorescence labelled SSR, new variety protection, variety identification

陶乃奇,张斌,刘信凯,周和达,钟乃盛,严丹峰,张敏,高继银,张文驹. 利用荧光标记SSR鉴别21个茶花新品种. 植物学报, 2019, 54(1): 37-45.

Naiqi Tao,Bin Zhang,Xinkai Liu,Heda Zhou,Naisheng Zhong,Danfeng Yan,Min Zhang,Jiyin Gao,Wenju Zhang. Identification of 21 New Camellia Hybrid Varieties by Fluorescence-labelled Simple Sequence Repeat Markers. Chinese Bulletin of Botany, 2019, 54(1): 37-45.

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链接本文: https://www.chinbullbotany.com/CN/10.11983/CBB18019

https://www.chinbullbotany.com/CN/Y2019/V54/I1/37

图/表 7

参考文献 38

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Cross combination No.	New variety No.	Parental types of cross combinations
ZH-01	SJ-01	Var. B (♀)×Var. A (♂)
	SJ-02	Var. B (♀)×Var. A (♂)
	SJ-03	Var. B (♀)×Var. A (♂)
	SJ-04	Var. B (♀)×Var. A (♂)
ZH-02	SJ-05	Var. C (♀)×Var. A (♂)
	SJ-06	Var. C (♀)×Var. A (♂)
	SJ-07	Var. C (♀)×Var. A (♂)
	SJ-08	Var. C (♀)×Var. A (♂)
ZH-03	SJ-09	Var. D (♀)×Var. A (♂)
	SJ-10	Var. D (♀)×Var. A (♂)
	SJ-11	Var. D (♀)×Var. A (♂)
ZH-04	SJ-12	Var. E (♀)×Var. A (♂)
ZH-05	SJ-13	Var. F (♀)×Var. A (♂)
ZH-06	SJ-14	Var. A (♀)×Var. G (♂)
	SJ-15	Var. A (♀)×Var. G (♂)
	SJ-16	Var. A (♀)×Var. G (♂)
ZH-07	SJ-17	Var. A (♀)×Var. H (♂)
ZH-07	SJ-18	Var. A (♀)×Var. H (♂)
ZH-08	SJ-19	Var. A (♀)×Var. I (♂)
ZH-09	SJ-20	Var. A (♀)×Var. J (♂)
ZH-10	SJ-21	Var. A (♀)×Var. K (♂)

Cross combination No.	New variety No.	Parental types of cross combinations
ZH-01	SJ-01	Var. B (♀)×Var. A (♂)
	SJ-02	Var. B (♀)×Var. A (♂)
	SJ-03	Var. B (♀)×Var. A (♂)
	SJ-04	Var. B (♀)×Var. A (♂)
ZH-02	SJ-05	Var. C (♀)×Var. A (♂)
	SJ-06	Var. C (♀)×Var. A (♂)
	SJ-07	Var. C (♀)×Var. A (♂)
	SJ-08	Var. C (♀)×Var. A (♂)
ZH-03	SJ-09	Var. D (♀)×Var. A (♂)
	SJ-10	Var. D (♀)×Var. A (♂)
	SJ-11	Var. D (♀)×Var. A (♂)
ZH-04	SJ-12	Var. E (♀)×Var. A (♂)
ZH-05	SJ-13	Var. F (♀)×Var. A (♂)
ZH-06	SJ-14	Var. A (♀)×Var. G (♂)
	SJ-15	Var. A (♀)×Var. G (♂)
	SJ-16	Var. A (♀)×Var. G (♂)
ZH-07	SJ-17	Var. A (♀)×Var. H (♂)
ZH-07	SJ-18	Var. A (♀)×Var. H (♂)
ZH-08	SJ-19	Var. A (♀)×Var. I (♂)
ZH-09	SJ-20	Var. A (♀)×Var. J (♂)
ZH-10	SJ-21	Var. A (♀)×Var. K (♂)

No.	Primer sequences (5′-3′)	Repeat motifs	Ta (°C)	References
478	F: CAACACCACCAACAAGA	(AAAGG)4	53	Liu et al., 2008
478	R: GATATGAGATCCGTCCC	(AAAGG)4	53	Liu et al., 2008
SSR2	F: TATTGCCTACGACCATTTCCA	(GA)14	56	Kaundun and Matsumoto, 2002
SSR2	R: TTTGAGTTCGTTGCCTTCTCT	(GA)14	56	Kaundun and Matsumoto, 2002
CamsinM11	F: GCATCATTCCACCACTCACC	(CA)12	60	Freeman et al., 2004
CamsinM11	R: GTCATCAAACCAGTGGCTCA	(CA)12	60	Freeman et al., 2004
CamSSR01	F: CCAACAAGAATCAGGAAGAG	(AAT)6	54	In this study
CamSSR01	R: ATCCAACGGTGGTAGACGAG	(AAT)6	54	In this study
CamSSR02	F: AGTTCCGCCTCCAGTTTGAC	(ACG)7	54	In this study
CamSSR02	R: GGACCGAGAGGTAACAGTGG	(ACG)7	54	In this study
CamSSR03	F: GCCACTACCCTCTTTACACC	(CAC)7	55	In this study
CamSSR03	R: TTCTCTTCCTCTTTCTTCCC	(CAC)7	55	In this study
CamSSR04	F: ATGTGTTGAGTAGCGAGCGT	(AT)10	56	In this study
CamSSR04	R: TTGTCCATCTTTATGTAGGG	(AT)10	56	In this study
CamSSR05	F: GCAAACACCAACTGATTACC	(TA)10	56	In this study
CamSSR05	R: TTCCATACAACTCAACCAAA	(TA)10	56	In this study
CamSSR06	F: GGTTTGGAAAAAGGACACGC	(GCC)7	58	In this study
CamSSR06	R: AATCTGCCTCTGGTAGTCCG	(GCC)7	58	In this study
CamSSR07	F: TCTCATCCCCATCTTTATCC	(TCC)7	58	In this study
CamSSR07	R: GTTCCCTGCTGCTGTTGTTA	(TCC)7	58	In this study
CamSSR08	F: TCACCAGTCACTTTCCCTCC	(AC)10	58	In this study
CamSSR08	R: CCACCAAAAGGCACAATACC	(AC)10	58	In this study
CamSSR09	F: CATCATCCATCAAACCGTCC	(AT)10	58	In this study
CamSSR09	R: GAAGGCACATTGGTTCTGGG	(AT)10	58	In this study

No.	Primer sequences (5′-3′)	Repeat motifs	Ta (°C)	References
478	F: CAACACCACCAACAAGA	(AAAGG)4	53	Liu et al., 2008
478	R: GATATGAGATCCGTCCC	(AAAGG)4	53	Liu et al., 2008
SSR2	F: TATTGCCTACGACCATTTCCA	(GA)14	56	Kaundun and Matsumoto, 2002
SSR2	R: TTTGAGTTCGTTGCCTTCTCT	(GA)14	56	Kaundun and Matsumoto, 2002
CamsinM11	F: GCATCATTCCACCACTCACC	(CA)12	60	Freeman et al., 2004
CamsinM11	R: GTCATCAAACCAGTGGCTCA	(CA)12	60	Freeman et al., 2004
CamSSR01	F: CCAACAAGAATCAGGAAGAG	(AAT)6	54	In this study
CamSSR01	R: ATCCAACGGTGGTAGACGAG	(AAT)6	54	In this study
CamSSR02	F: AGTTCCGCCTCCAGTTTGAC	(ACG)7	54	In this study
CamSSR02	R: GGACCGAGAGGTAACAGTGG	(ACG)7	54	In this study
CamSSR03	F: GCCACTACCCTCTTTACACC	(CAC)7	55	In this study
CamSSR03	R: TTCTCTTCCTCTTTCTTCCC	(CAC)7	55	In this study
CamSSR04	F: ATGTGTTGAGTAGCGAGCGT	(AT)10	56	In this study
CamSSR04	R: TTGTCCATCTTTATGTAGGG	(AT)10	56	In this study
CamSSR05	F: GCAAACACCAACTGATTACC	(TA)10	56	In this study
CamSSR05	R: TTCCATACAACTCAACCAAA	(TA)10	56	In this study
CamSSR06	F: GGTTTGGAAAAAGGACACGC	(GCC)7	58	In this study
CamSSR06	R: AATCTGCCTCTGGTAGTCCG	(GCC)7	58	In this study
CamSSR07	F: TCTCATCCCCATCTTTATCC	(TCC)7	58	In this study
CamSSR07	R: GTTCCCTGCTGCTGTTGTTA	(TCC)7	58	In this study
CamSSR08	F: TCACCAGTCACTTTCCCTCC	(AC)10	58	In this study
CamSSR08	R: CCACCAAAAGGCACAATACC	(AC)10	58	In this study
CamSSR09	F: CATCATCCATCAAACCGTCC	(AT)10	58	In this study
CamSSR09	R: GAAGGCACATTGGTTCTGGG	(AT)10	58	In this study

Primers	Na	Ne	PIC
478	7.00	4.12	0.72
SSR2	6.00	2.63	0.58
CamsinM11	4.00	1.85	0.43
CamSSR01	3.00	1.48	0.29
CamSSR02	4.00	2.97	0.62
CamSSR03	6.00	4.96	0.77
CamSSR04	6.00	2.99	0.62
CamSSR05	4.00	2.82	0.58
CamSSR06	4.00	1.76	0.41
CamSSR07	4.00	1.58	0.35
CamSSR08	6.00	4.96	0.77
CamSSR09	5.00	2.32	0.49
(mean)	4.92	2.87	0.55

利用荧光标记SSR鉴别21个茶花新品种

Identification of 21 New Camellia Hybrid Varieties by Fluorescence-labelled Simple Sequence Repeat Markers

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Code of pattern	Cam- SSR08	Cam- SSR04	478	SSR2	Cam- SSR03	Cam- SSR09	Cam- SSR06	Cam- sinM11	Cam- SSR01	Cam- SSR07	Cam- SSR05	Cam- SSR02
1	125/131	165/167	113/115	217	278/313	209/215	176/185	171	188/200	206	234/240	271/274
2	125/135	165/171	113/129	217/219	278/321	211/217	179/182	173	195	206/212	234/242	271/277
3	127	167	115	217/225	278/322	213/215	179/185	177	195/200	206/215	236/240	271/283
4	127/131	167/171	115/129	217/227	278/323	215	182/185	181	200	206/221
5	129	167/173	117/129	217/231	307/321	215/217	185
6	129/131	169/171	121/129	217/235	307/322	217
7	129/133	171	123/129	219/235	307/323
8	129/135	171/173	123/133
9	131/133	171/175
A	133
B	133/135

No.	Molecular identity code	No.	Molecular identity code
SJ-01	192315514322	SJ-12	393121414432
SJ-02	291315514322	SJ-13	485225334121
SJ-03	122516514322	SJ-14	546224111121
SJ-04	897336114322	SJ-15	545254111121
SJ-05	975265414113	SJ-16	646254114121
SJ-06	913235513113	SJ-17	353661513121
SJ-07	A73135522123	SJ-18	445664323113
SJ-08	A35165424121	SJ-19	934175513111
SJ-09	973663214212	SJ-20	563424544123
SJ-10	972646514212	SJ-21	748772514411
SJ-11	B61664544212

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[2]	徐颖, 徐晶, 高继银, 张文驹. 山茶属植物ITS的多态性——一个广泛逃离一致性进化的实例[J]. 植物学报, 2011, 46(2): 162-169.
[3]	梁明山曾宇周翔侯留记李霞. 遗传标记及其在作物品种鉴定中的应用[J]. 植物学报, 2001, 18(03): 257-265.