基于SSR标记的山茶属位点组合品种鉴别能力分析评价

doi:10.11707/j.1001-7488.LYKX20220787

摘要/Abstract

摘要：

目的: 筛选多态性高、通用性强等的SSR位点，评估筛选所得SSR位点的组合在山茶属品种中的鉴别能力；在保持最大鉴别能力的基础上，确定可用于山茶属品种鉴别所需的最少SSR位点的组合，提高品种鉴别效率。方法: 本研究以111份山茶属品种为材料，结合凝胶电泳、荧光毛细管电泳、位点多态性分析和位点组合品种鉴别力（VDP）分析等方法，筛选评价SSR位点并对SSR位点组合的品种鉴别力进行分析与评价。结果: 本研究筛选获得17个SSR位点，占筛选位点总数的30.9%，在111份山茶属品种中，共检测到166个等位基因，单位点基因型数量范围为6～35，均值为23.353个，观测等位基因数（N_a）范围为3～15，均值为9.765个，多态性信息含量（PIC）的范围为0.447～0.835，均值为0.696，位点缺失率的范围为0～4.50%，均值为0.90%。当差异位点数=1时，17个SSR位点在全部品种（系）、红山茶组品种和金花茶组无性系中达到的R-VDP最大值均为1，可将SSR位点数量分别缩减到11、10、5个，分别占筛选所得位点数的64.7%，58.8%和29.4%；当差异位点数=2时，17个SSR位点在全部品种（系）、红山茶组品种和金花茶组无性系中达到的R-VDP最大值分别为0.964，0.952和1.000，可将SSR位点数量分别缩减到12、12、和7个，分别占筛选所得位点数的70.6%、70.6%和41.2%；当差异位点数=3时，17个SSR位点在全部品种（系）、红山茶组品种和金花茶组无性系中达到的R-VDP最大值分别为0.946，0.929，1.000，可将SSR位点数量分别缩减到8、7和8个，分别占筛选所得位点数的47.1%、41.2%和47.1%。当差异位点数≤6时，17个SSR位点组合对山茶属测试品种（系）群的R-VDP最大值可保持相对稳定的水平。结论: 本研究筛选验证获得17个多态性高、通用性强等的SSR位点；这些位点的组合在山茶属品种（系）群中均表现出良好的品种鉴别能力；确定了山茶属不同类群品种（系）鉴别所需的最少SSR位点的组合，构建了一套山茶属SSR位点组合品种鉴别能力分析评价的体系，为山茶属品种鉴别及相关标准的制定等奠定了基础。

关键词: 山茶属, SSR标记, 位点组合, 品种鉴别能力, VDPtools

Abstract:

Objective: The paper aims to screen SSR loci with high polymorphism and strong universality in Camellia, evaluate the variety discrimination power (VDP) of different SSR loci combinations and determine the minimum numbers of SSR loci in combination for variety discrimination at different Camellia groups, so as to improve the efficiency of variety discrimination in practice. Method: In this study, a total of 111 Camellia varieties were used to screen and evaluate SSR loci and the variety discrimination power of combinations of SSR loci by combining the methods of gel electrophoresis, fluorescent capillary electrophoresis, loci polymorphism analysis and loci combination variety discrimination power (VDP) analysis. Result: A total of 17 SSR loci were screened and obtained, accounting for 30.9% of the total number of selected loci. In 111 Camellia varieties, 166 alleles were detected with the 17 SSR loci. The number of genotypes per locus ranged from 6 to 35, with an average of 23.353. The observed number of alleles (N_a) ranged from 3 to 15, with a mean of 9.765. The polymorphic information content (PIC) ranged from 0.447 to 0.835, with a mean of 0.696. The range of missing data rate of loci was 0–4.50%, with a mean of 0.90%. When the number of differential loci=1, the maximum R-VDP of 17 SSR loci in all varieties (clones), Sect. Camellia varieties and Sect. Chrysantha clones were all 1, and the number of SSR loci was able to be reduced to 11, 10 and 5, respectively accounting for 64.7%, 58.8% and 29.4% of the screened loci in the different groups. When the number of differential loci=2, the maximum R-VDP values of 17 SSR loci in all varieties (clones), Sect. Camellia varieties and Sect. Chrysantha clones were 0.964, 0.952 and 1.000, respectively, and the number of SSR loci was able to be reduced to 12, 12 and 7, respectively accounting for 70.6%, 70.6% and 41.2% of the screened loci in the different groups. When the number of differential loci=3, the maximum R-VDP values of 17 SSR loci in all varieties (clones), Sect. Camellia varieties and Sect. Chrysantha clones were 0.946, 0.929, and 1.000, respectively, and the number of SSR loci was able to be reduced to 8, 7 and 8, respectively, accounting for 47.1%, 41.2% and 47.1% of the screened loci in the different groups. When the number of differential loci was ≤ 6, the maximum R-VDP of 17 SSR loci combinations for the tested varieties (clones) of Camellia could maintain a relatively stable level. Conclusion: In this study, 17 SSR loci with high polymorphism and strong universality have been screened and verified. The combinations of these loci show well variety discrimination power in the tested Camellia groups. The minimum numbers of SSR loci in combinations for variety discrimination have been determined in different variety (clones) groups of Camellia, and a set of analyses and evaluation system for variety discrimination power of combinations of SSR loci in Camellia have been constructed. These results provide useful information and tools for variety identification and development of related standards.

Key words: Camellia, SSR marker, combination of SSR loci, variety discrimination power, VDPtools

中图分类号:

李丰钰,黄平,郑勇奇,李长红,张雨婷,薛克娜,宗亦臣,赵鸿杰. 基于SSR标记的山茶属位点组合品种鉴别能力分析评价[J]. 林业科学, 2023, 59(8): 74-84.

Fengyu Li,Ping Huang,Yongqi Zheng,Changhong Li,Yuting Zhang,Kena Xue,Yichen Zong,Hongjie Zhao. Analysis and Evaluation of Variety Discrimination Power among Genus Camellia with Loci Combinations Selected by Using SSR Markers[J]. Scientia Silvae Sinicae, 2023, 59(8): 74-84.

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位点名称 Name of loci	基因型数 Number of genotypes	多态性信息含量 PIC	等位基因数 N_a	数据缺失率 Missing data rates (%)
p1	28	0.835	10	2.70
CamSSR08	27	0.820	9	0.00
p85	33	0.816	12	4.50
p40	22	0.811	10	0.00
CamSSR09	23	0.798	9	0.00
p12	30	0.789	12	1.80
478	34	0.781	13	0.90
p78	24	0.727	9	0.00
TUGM78	35	0.707	14	0.00
P06	33	0.697	15	0.00
p24	22	0.696	13	0.00
CSSR5	20	0.679	9	3.60
CamSSR06	20	0.668	8	0.00
p41	6	0.534	3	0.00
CamSSR02	12	0.516	8	0.90
A25	11	0.513	5	0.90
CSSR35	17	0.447	7	0.00
平均Mean	23.353	0.696	9.765	0.90

位点名称 Name of loci	正向引物序列 Forward primers (5'—3')	反向引物序列 Reverse primers (5'—3')	基序 Motif	产物大小 Product size	退火温度 Annealing temperature (T_m)/℃	参考文献 Reference
A25	GGCTTGGACACTTGGTTAGA	AAGATGGATTAGGGTAGGAT	—	186～207	48	张亚利等，2016
TUGM78	CACCGCTTGACTAAAATGG	AAACTATCAACCGTATGGGC	—	101～161	55	张旻桓等，2018
P06	CAGGGTTGCAAGAAGTACCG	ATCAACCGTATGGGCAAAAG	—	118～178	57	张旻桓等，2018
478	CAACACCACCAACAAGA	GATATGAGATCCGTCCC	—	111～137	53	陶乃奇等，2019
CamSSR08	TCACCAGTCACTTTCCCTCC	CCACCAAAAGGCACAATACC	(AC)₁₀	120～138	58	陶乃奇等，2019
CamSSR09	CATCATCCATCAAACCGTCC	GAAGGCACATTGGTTCTGGG	(AT)₁₀	205～221	58	陶乃奇等，2019
CamSSR06	GGTTTGGAAAAAGGACACGC	AATCTGCCTCTGGTAGTCCG	(GCC)₇	176～197	58	陶乃奇等，2019
CamSSR02	AGTTCCGCCTCCAGTTTGAC	GGACCGAGAGGTAACAGTGG	(ACG)₇	263～287	54	陶乃奇等，2019
p85	GCGTTGATCATGGTTTATCG	CCGTTGATCCCTTCGACTTA	(ATG)₅	278～329	55	潘丽芹等，2019
p12	TCAAAAGAGACCTTGGGCTG	GGGGACTTCCGATAACACAA	—	270～306	55	潘丽芹等，2019
p1	CTTCTTCTCGATCCACAGCC	CGATCTCCTCCGTAACAAGC	(GAG)₅	186～222	55	潘丽芹等，2019
p78	TCCCCATGTAGACTCTTCCG	AAGACATGTTCGGTTCCGTC	(CCG)₅	255～282	55	潘丽芹等，2019
p24	GTGAAACAAAGCCGGAGAGT	ACCTGGTTCAATCTATGGCG	(GA)₇	219～249	55	潘丽芹等，2019
p40	AACAATACCCGACTCCTCCC	CCTATGGCGAGACGTTCAAT	(CAT)₆	283～325	55	潘丽芹等，2019
p41	AATAGCACGGTAATCACGGC	GAATTTTCTGGGCCATCTGA	(CAT)₇	233～239	55	潘丽芹等，2019
CSSR5	GCTGTAGGCGAACATGAA	CACTTCCACTTCCATATCCA	(GGTGCT)₆	180～231	55	Tong et al., 2020
CSSR35	ATCGCAGACAACAAGAAGA	GGAGGAGATCGTGATGAAG	(TGA)₆	114～132	55	Tong et al., 2020

差异位点数 Number of differential loci	山茶属品种（系）群 Variety groups of Camellia	R-VDP最大值 Maximum R-VDP	所需最少位点数（Minimum number of loci required）	位点组合 Combination of SSR Loci
1	全部品种（系） All varieties (clones)	1	11	p1、CamSSR08、 p85、p40、CamSSR09、TUGM78、P06、CSSR5、CamSSR06、 p41、CamSSR02
	红山茶组品种 Sect. Camellia varieties	1	10	p1、CamSSR08、 p85、p40、TUGM78、P06、CSSR5、CamSSR06、p41、CamSSR02
	金花茶组无性系 Sect. Chrysantha clones	1	5	p1、CamSSR08、 p85、p40、CamSSR09
2	全部品种（系） All varieties (clones)	0.964	12	p1、CamSSR08、 p85、p40、CamSSR09、 p12、478、p78、TUGM78、P06、CSSR5、CamSSR02
	红山茶组品种 Sect. Camellia varieties	0.952	12	p1、CamSSR08、 p85、p40、CamSSR09、 p12、478、p78、TUGM78、P06、CSSR5、CamSSR02
	金花茶组无性系 Sect. Chrysantha clones	1	7	p1、CamSSR08、 p85、p40、CamSSR09、 p12、478
3	全部品种（系） All varieties (clones)	0.946	8	p1、CamSSR08、 p85、p40、CamSSR09、 p12、478、p78
	红山茶组品种 Sect. Camellia varieties	0.929	7	p1、CamSSR08、 p85、p40、CamSSR09、 p12、p78
	金花茶组无性系 Sect. Chrysantha clones	1	8	p1、CamSSR08、 p85、p40、CamSSR09、 p12、478、p78

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