基于荧光SSR标记的接骨木种质资源遗传多样性分析

doi:10.12302/j.issn.1000-2006.202305037

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南京林业大学学报（自然科学版） ›› 2025, Vol. 49 ›› Issue (2): 75-82.doi: 10.12302/j.issn.1000-2006.202305037

基于荧光SSR标记的接骨木种质资源遗传多样性分析

姚俊修¹(), 任飞¹, 王因花¹, 李庆华¹, 燕丽萍¹, 郑岩², 吴德军¹^,^*()

1.山东省林业科学研究院,山东省生态林草遗传育种重点实验室,山东济南 250014
2.山东高速鸿林工程技术有限公司,山东济南 250101

收稿日期:2023-05-31 接受日期:2024-09-29 出版日期:2025-03-30 发布日期:2025-03-28
通讯作者: *吴德军(sdlky412@163.com),研究员。
作者简介:
姚俊修(yjx95289528@163.com),高级工程师。
基金资助:
山东省重点研发计划(农业良种工程)(2020LZGC0905);中央财政林业改革发展资金项目(鲁[2024]TG05号);山东高速集团科技创新项目(SDGSHL-2024-009)

Genetic diversity of germplasm resources of Sambucus based on SSR fluorescent marker

YAO Junxiu¹(), REN Fei¹, WANG Yinhua¹, LI Qinghua¹, YAN Liping¹, ZHENG Yan², WU Dejun¹^,^*()

1. Shandong Academy of Forestry, Shandong Provincial Key Laboratory of Ecological Forest and Grassland Genetics and Breeding, Ji’nan 250014, China
2. Shandong Expressway Honglin Engineering Technology Co., Ltd., Ji’nan 250101, China

Received:2023-05-31 Accepted:2024-09-29 Online:2025-03-30 Published:2025-03-28

摘要/Abstract

摘要：

【目的】以收集的245份接骨木属(Sambucus)种质资源和60份接骨木杂交种质为材料,基于SSR分子标记对不同种源接骨木的遗传多样性及亲缘关系进行分析,为接骨木种质资源的合理保护、评价与利用提供科学依据。【方法】以11个种源的305个体为研究对象,根据转录组测序结果,开发100对SSR引物,对8个不同种接骨木个体进行扩增筛选,获得13对多态性较好的SSR引物,使用Popgene、Genemarker、GeneALeX和NTSYSpc等软件,对接骨木种质资源及杂交子代的遗传多样性、遗传距离、不同种源主坐标、UPGMA关系及进化树等进行分析。【结果】利用13对荧光引物对305个体进行扩增,检测到等位基因总数为162,不同位点等位基因变异幅度为4~34个,平均为12.5个;有效等位基因总数(N_e)为50.408个,变异幅度为1.066 1~11.070 2个,平均为3.878;平均观察杂合度(H_o)为0.331 5,平均期望杂合度(H_e)为0.521 5,平均Nei’s遗传距离为0.520 4,平均多态性信息含量为0.496 4,表明遗传多样性较高;42号位点H_o/H_e大于1,表明该位点杂合度高,种群内遗传变异程度较大。不同种源参试样本中H_o为0.208(杂交组合1)~0.486(长白县),平均H_o为0.372。H_e为0.242(杂交组合1)~0.552(大庆),平均H_e为0.434,其中,长白县、朝鲜种源及杂交组合2的H_o/H_e均大于1,表明这3个种源/杂交组合内群体杂合度较高,种群内遗传变异程度也较大。主坐标分析表明:遗传变异主要存在于个体间,占80%,而种源间遗传变异仅占20%,可能由于不同地区的栽培种源来源于相同省域,造成不同种源间变异较小;Nei’s遗传距离和聚类结果表明,种源/杂交组合间的遗传距离变化范围为0.023~0.637,山东青州仰天山天然群体和吉林长白县的遗传分化差异最大;内蒙赤峰和新疆阿勒泰的遗传分化差异最小;聚类可分为3大类群,同一种源个体并未完全聚到相同分支,可能原因是遗传变异或相互引种造成的。【结论】接骨木种质资源具有较高的遗传多样性,在进行接骨木遗传改良时,应首先进行种源内选择育种及单株改良的育种方法,其次可考虑种源间研究。研究结论为接骨木种质资源精准鉴定、核心种质构建及新品种创制等奠定基础。

关键词: 荧光SSR标记, 接骨木, 种质资源, 遗传多样性

Abstract:

【Objective】This study analyzed the genetic diversity and genetic relationship of Sambucus williamsii from different provenances based on SSR molecular markers, to provide a scientific basis for the rational protection, evaluation, and utilization of S. williamsii germplasm resources.【Method】Using 245 samples of Sambucus germplasm resources and 60 samples of Sambucus hybrid germplasm as the test materials, 100 pairs of SSR primers were developed based on the transcriptome sequencing results. Through the amplification and screening of eight different individuals of Sambucus species, 13 pairs of SSR primers with good polymorphism were obtained. The genetic diversity, genetic distance, principal coordinates of different species origins, UPGMA relationships, and evolutionary trees of the Sambucus germplasm resources and their hybrid offspring were examined using the Popgene, Genemarker, GeneALeX and NTSYSpc software packages.【Result】We employed 13 pairs of fluorescent primers to amplify 305 individuals, and 162 alleles were detected. The variation range of alleles at different loci was determined as 4-34, with an average of 12.5 alleles. The total number of effective number of allele ( N_e) was 50.408, with a variation range of 1.066 1-11.070 2. The average N_e, abserved heterozygosity (H_o), and expected heterozygosity (H_e) values were 3.878, 0.331 5 and 0.521 5, respectively. Moreover, we determined the average Nei’s genetic distance (Nei) and polymorphism information content (PIC) values of 0.520 4 and 0.496 4, respectively. These results suggest a high genetic diversity. The H_o/H_e ratio at site 42 was greater than 1, indicating high degrees of heterozygosity and genetic variation within the species. Among the tested samples from different species origins, H_o ranged from 0.208 (hybrid combination 1) to 0.486 (Changbai County), with an average H_o value of 0.372. H_e ranged from 0.242 (hybrid combination 1) to 0.552 (Daqing), with an average H_e value of 0.434. Notably, the H_o/H_e ratios for the species origins (or hybrid combinations) of Changbai County, North Korea provenances, and hybrid combination 2 were all greater than 1, indicating that these three species origins (or hybrid combinations) had higher population heterozygosity and greater degrees of genetic variation within the populations. Principal coordinate analysis revealed that the majority of genetic variation, accounting for 80%, was found among individuals, while genetic variation among species origins accounted for only 20%. This may be because the cultivated varieties from different regions originated from the same provincial area, leading to smaller genetic variation among species of different origins. The genetic distance of Nei among species origins (or hybrid combinations) ranged from 0.023 to 0.637. The greatest genetic differentiation was observed between the natural populations of Yangtianshan in Qingzhou, Shandong, and Changbai County, Jilin, and the lowest between the populations of Chifeng, Inner Mongolia, and Altay, Xinjiang. The clustering could be divided into three groups, and the same source individuals were not completely clustered into the same branch, which may be caused by genetic variation or mutual introduction.【Conclusion】We demonstrated the high genetic diversity of the germplasm resources of S. williamsii. To achieve genetic improvement, intra-provenance selection and single plant improvement should initially be performed, followed by inter-provenance selection. This study provides a theoretical basis for the precise identification of S. germplasm resources, the construction of core germplasms, and the creation of new varieties.

Key words: fluorescent SSR marker, Sambucus williamsii, germplasm resources, genetic diversity

中图分类号:

S722.3

姚俊修,任飞,王因花,等. 基于荧光SSR标记的接骨木种质资源遗传多样性分析[J]. 南京林业大学学报（自然科学版）, 2025, 49(2): 75-82.

YAO Junxiu, REN Fei, WANG Yinhua, LI Qinghua, YAN Liping, ZHENG Yan, WU Dejun. Genetic diversity of germplasm resources of Sambucus based on SSR fluorescent marker[J].Journal of Nanjing Forestry University (Natural Science Edition), 2025, 49(2): 75-82.DOI: 10.12302/j.issn.1000-2006.202305037.

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SSR 编号 SSR code	正向引物序列 forward primer sequence (5'—3')	反向引物序列 reverse primer sequence (5'—3')	产物大小/bp product size
3	AAAAGGGTTTGGCTTGGGCA	GTCACCCCCACAAAGCCTAG	258
37	AACCTGGTGTCCACGAGGTC	TTGTTCTAGCCCCCACTCCA	126
39	AACGTGAGCAACAACCCCAA	TCAATTGTCGGGCTTCACTGT	216
42	AACTGTAGCGAGAGGCCAAA	TATACCCGAGCTCTGGGGAA	210
43	AACTTCAAACTGCATCGCCG	AGCGTGATAGCAGGGAAAGA	148
48	AAGACCCTGTATGGTTGGGG	CCTGTACTCGTGGAGCCTTT	240
55	AAGCGCGTCACATTCCCATA	AGACACTGCGCACAAGTTGT	279
63	AAGGCTATCCTAGTGAGCGG	TTCCGTAGACGCACGTATTCC	256
67	AAGTGGGCAGAACCAAGTGA	GAGCTGTATCTGGTCGAGGG	236
87	AATTGCGTGCCCTCGAAAAC	TGTTTCAGCGACGCAAAGGA	268
90	ACAAAAACCTCACGGCTCGT	CCAGTGATCCTTATGTGCGC	243
92	ACAAAGGGTAACATGCACTTTTCT	TGCAACACCACAAACTGTCCT	213
97	ACAACTAGCTTGGGGAGTGG	ACAACCACAATTGCCGTTAGC	192

位点 locus	等位基因数 allele number (N_a)	有效等位基因数 effective number of allele(N_e)	Shannon 信息指数 Shannon information index(I)	观测杂合度 observed heterozygosity (H_o)	期望杂合度 expected heterozygosity (H_e)	Nei’s遗传距离 Nei’s genetic distance(Nei)	多态性信息含量 polymorphism information content(PIC)
3	15	5.877 0	1.990 2	0.496 0	0.831 5	0.829 8	0.809 3
67	34	11.070 2	2.838 3	0.682 5	0.911 5	0.909 7	0.903 8
97	11	1.677 9	1.009 0	0.250 0	0.404 8	0.404 0	0.392 9
87	4	1.520 4	0.596 3	0.242 1	0.343 0	0.342 3	0.296 0
39	19	7.288 8	2.272 3	0.638 9	0.864 5	0.862 8	0.848 4
43	4	1.989 3	0.766 2	0.154 8	0.498 3	0.497 3	0.392 2
55	12	3.368 3	1.419 1	0.115 1	0.704 5	0.703 1	0.649 9
48	6	1.210 9	0.418 9	0.115 1	0.174 5	0.174 1	0.168 2
42	6	1.277 8	0.454 3	0.222 2	0.217 9	0.217 4	0.202 0
90	24	7.052 1	2.351 5	0.730 2	0.859 9	0.858 2	0.844 5
92	19	5.935 5	2.111 1	0.623 0	0.833 2	0.831 5	0.812 2
37	4	1.066 1	0.167 1	0.023 8	0.062 1	0.062 0	0.061 0
63	4	1.079 2	0.198 8	0.015 9	0.073 6	0.073 4	0.072 4
平均mean	12.46	3.878 0	1.276 4	0.331 5	0.521 5	0.520 4	0.496 4

种源 provenance	编号 code	取样数 sample number	N_a	N_e	I	H_o	H_e	固定指数 fixed index(F)
哈尔滨 Harbin	H	31	7.286	3.140	1.099	0.346	0.468	0.323
新疆(阿勒泰) Xinjiang (Altay Mountains)	XJ	36	7.214	3.346	1.180	0.428	0.510	0.275
长白县(吉林) Changbai County (Jilin)	CBX	5	3.500	2.723	0.865	0.486	0.440	-0.033
朝鲜普通 North Korea normal	CX	8	2.357	2.153	0.648	0.391	0.381	0.006
朝鲜多果 Korean multi-fruit	CXDG	5	3.000	2.279	0.759	0.457	0.409	-0.068
赤峰(内蒙古) Chifeng (Inner Mogolia)	CF	24	6.643	3.128	1.150	0.396	0.501	0.320
大庆(黑龙江) Daqing (Helongjiang)	DQ	11	5.786	3.446	1.218	0.364	0.552	0.431
呼和浩特 Hohhot	HS	21	5.929	3.311	1.088	0.398	0.483	0.142
集安(吉林) Ji’an (Jilin)	JA	24	4.929	2.662	0.918	0.384	0.437	0.123
通化(吉林) Tonghua (Jilin)	TH	26	6.714	3.949	1.196	0.434	0.523	0.170
珲春(吉林) Hunchun (Jilin)	HC	21	5.571	3.265	1.063	0.375	0.484	0.194
杂交组合1(山东)(Shandong) hybrid combination 1(Shandong)	济8×济4 [HY(8×4)]	28	2.714	1.543	0.437	0.208	0.242	0.499
杂交组合2(山东) hybrid combination 2	济8×济5 [HY(8×5)]	32	2.429	1.718	0.511	0.325	0.314	-0.011
青州(山东仰天山) Qingzhou (Yangtian Mountain’Shandong)	YTS	28	2.643	1.780	0.543	0.305	0.306	0.034
平均 mean		21.8	4.901	2.782	0.917	0.372	0.434	0.190

种源 provenance	H	XJ	CBX	CX	CXDG	CF	DQ	HS	JA	TH	HC	HY (8×4)	HY (8×5)
XJ	0.024
CBX	0.139	0.159
CX	0.104	0.097	0.190
CXDG	0.107	0.075	0.222	0.167
CF	0.034	0.023	0.157	0.119	0.079
DQ	0.056	0.063	0.188	0.126	0.137	0.076
HS	0.160	0.180	0.255	0.225	0.272	0.192	0.091
JA	0.140	0.171	0.238	0.247	0.250	0.164	0.112	0.052
TH	0.098	0.120	0.181	0.165	0.230	0.130	0.079	0.050	0.073
HC	0.179	0.194	0.299	0.234	0.263	0.212	0.101	0.040	0.061	0.071
HY(8×4)	0.352	0.326	0.455	0.464	0.343	0.372	0.356	0.435	0.408	0.402	0.422
HY(8×5)	0.351	0.311	0.490	0.423	0.418	0.367	0.350	0.402	0.390	0.368	0.389	0.111
YTS	0.489	0.453	0.637	0.518	0.418	0.477	0.478	0.525	0.524	0.557	0.515	0.341	0.340

基于荧光SSR标记的接骨木种质资源遗传多样性分析

Genetic diversity of germplasm resources of Sambucus based on SSR fluorescent marker

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类群 group	哈尔滨 H	新疆 XJ	长白县 CBX	朝鲜普通 CX	赤峰 CF	大庆 DQ	呼和浩特 HS	集安 JA	通化 TH	珲春 HC	青州 YTS	杂交组合1 HY (8×4)	杂交组合2 HY (8×5)	朝鲜多果 CXDG	总计 total
1	1	2	0	0	2	0	0	0	0	0	0	0	0	0	5
2	5	4	0	1	3	3	5	5	3	7	6	12	6	1	61
3	25	30	5	2	19	8	16	19	23	14	32	20	22	4	239
总计total	31	36	5	3	24	11	21	24	26	21	38	32	28	5	305

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