毛白杨微核心种质构建方法的探�?/title>

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doi:10.12171/j.1000-1522.20210150

1.
山东省林业科学研究院，山东省林木遗传改良重点实验室，山东济南 250014
2.
山东水发旅游开发有限公司，山东济南 250101
3.
山东省林草种质资源中心，山东济南 250014
4.
山东省国土空间规划院，山� 济南 250014
5.
冠县国有毛白杨林场，山东聊城 252000

基金项目:山东省重点研发计划（2021SFGC0205），林木遗传育种国家重点实验室开放基金项目（K2020203），林业科技发展项目（KJZXZZ2019015、KJZXSA202003（�/div>

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作者简今�
毛秀红，博士，正高级工程师。主要研究方向：林木遗传育种。Email９�a href="//www.inggristalk.com/j/article/doi/10.12171/mailto:xiuhongmao@163.com">xiuhongmao@163.com　地址�?50014 山东省济南市历下区文化东�?2号山东省林业科学研究陡�/p>

责任作耄�
臧真荣，正高级工程师。主要研究方向：林木遗传育种。Email９�a href="//www.inggristalk.com/j/article/doi/10.12171/mailto:mollion@126.com">mollion@126.com　地址：同三�/span>
李善文，博士，研究员。主要研究方向：林木遗传育种。Email９�a href="//www.inggristalk.com/j/article/doi/10.12171/mailto:lishanwen66@163.com">lishanwen66@163.com　地址：同三�/span>

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出版历程
- 收稿日期:2021-04-26
- 修回日期:2021-07-12
- 网络出版日期:2022-11-19
- 刊出日期:2023-02-25

Study on construction method of microcore germplasm ofPopulus tomentosa

1.
Shandong Academy of Forestry, Shandong Province Key Laboratory of Forestry Tree Genetic Improvement, Jinan 250014, Shandong, China
2.
Shandong Shuifa Tourism Development Co. Ltd, Jinan 250101, Shandong, China
3.
Shandong Forest and Grass Germplasm Resources Center, Jinan 250014, Shandong, China
4.
Shandong Province Institute of Territorial Space Planning, Jinan 250014, Shandong, China
5.
State-Owned Chinese White Poplar Forest Farm in Guanxian County, Guanxian 252500, Shandong, China

摘要

摘要: 目的探讨构建微核心种质的方法，旨在为精准选择毛白杨杂交亲本或研究材料提供科学依据，为其他物种构建微核心种质提供参考、�/sec> 方法本研究利用荧光SSR分子标记�?72份毛白杨样本进行分子基因型检测，计算每个样本对总体遗传多样性的贡献值，从大到小全部排序，计算新排序的前25%�?0%�?5%�?0%�?%�?.5%�?%�?.5%�?个取样比例的平均有效等位基因数（Ne）、平均Shannon信息指数＇�i>I），平均期望杂合度（He）和平均多态信息指数（PIC）值等，分析微核心种质的代表性，通过与前面的取样比例以及原始种质的相应参数进行比较，确定合适的微核心种质取样比例。利�?i>t检验进行统计学验证。根据所有引物的峰值，构建每份微核心种质的指纹图谱。基于指纹图谱的差异，分析挖掘特异等位基因、�/sec> 结果�?）当取样比例�?5%逐渐降为2%时，Ne、He和PIC�?个重要的遗传多样性参数分别逐渐达到最大值，耋�i>I在取样比例为10%时达到最大值。（2）当取样比例�?%时，Ne�?i>I、He和PIC值分别是3.513�?.254�?.643�?.597，均大于272份原始种质的相应�?.075�?.825�?.432�?.364，表明构建的微核心种质有效去除了遗传冗余，具有丰富的遗传多样性。（3（�i>t检验结果表明：所构建的微核心种质与原始种质的遗传多样性无显著差异，具有可靠的代表性，可以作为微核心种质。（4）不�?0%�?%还是2%取样比例，杂交种质所占比例均大于50%。（5�?18号样品在11号位点拥有特异等位基因，261号样品在16号位点拥有特异等位基因，263号样品在7号位点拥有特异等位基因、�/sec> 结论毛白杨微核心种质最为科学、可信、简约和高效的取样比例为2% ~ 10%，其中最精准的取样比例为2%。如果种质资源数目较大，可以根据遗传多样性适当降低取样比例；如果基数较小，可以适当升高。建议以后构建核心种质或者微核心种质时，不要先人为进行分组，而是先根据每个样品对总体遗传多样性的贡献从大到小排序。本研究从分子水平证明杂交种质在种质资源保存和林木遗传育种中占有重要地位，将为其他物种微核心种质构建提供参考、�/sec>

关键诌�

毛白�?/a> /

SSR分子标记/
微核心种�?/a> /

遗传多样�?/a> /

指纹图谱/
特异等位基因

Abstract: ObjectiveThe method of constructing microcore germplasm was discussed to provide scientific basis for precise selection of hybrid parents or research materials of P. tomentosa, and to provide reference for other species to construct microcore germplasm. MethodIn this study, molecular genotypes of 272 P. tomentosasamples were detected by fluorescent SSR molecular markers. The contribution value of each sample to the overall genetic diversity was calculated and sorted from large to small. Then, the representativity of microcore germplasm was analyzed and the appropriate sampling proportion was determined by comparing the average effective number of alleles Ne, the average Shannon’s information index I, the average expected heterogenicity He and the average polymorphic information content PIC values obtained by sampling proportion of top 25%, 20%, 15%, 10%, 5%, 2.5%, 2% and 1.5%. Finally, ttest was used for statistical verification. According to the band-shaped results of all SSR primers, fingerprints of each microcore germplasm were formed. According to the difference of fingerprint, the specific alleles were analyzed and excavated. ResultWhen the sampling proportion gradually decreased from 25% to 2%, three important genetic diversity parameters of Ne, He and PIC gradually reached the maximum values, respectively, while Ireached the maximum value when the sampling proportion was 10%. When the sampling ratio was 2%, the values of Ne, I, He and PIC were 3.513, 1.254, 0.643 and 0.597, respectively, which were all greater than 2.075, 0.825, 0.432 and 0.364 of the original germplasm, indicating that we had effectively removed the genetic redundancy, and the constructed microcore germplasm had abundant genetic diversity. The ttest results showed that there was no significant difference in genetic diversity between the constructed microcore germplasm and the original germplasm, which was reliable and representative and could be used as the microcore germplasm. The proportion of hybrid germplasm was more than 50% regardless of the sampling proportion of 10%, 5% or 2%. Sample 218 had a specific allele at locus 11, sample 261 had a specific allele at locus 16, and sample 263 had a specific allele at locus 7. ConclusionWhen the sampling ratio of microcore germplasm of poplar is 2%�?0%, it is the most scientific, reliable, simple and efficient, among which the most accurate sampling ratio is 2%. If the number of germplasm resources is large, it can be appropriately reduced according to the genetic diversity, and on the contrary, it can be increased appropriately. It is suggested that when constructing core germplasm or microcore germplasm in the future, rather than grouping, each sample is firstly ranked from largest to smallest according to its contribution to overall genetic diversity. It is proved from the molecular level that hybrid germplasm plays an important role in the conservation of germplasm resources and the genetic breeding of trees. At the same time, this study also provides reference for the construction of microcore germplasm of similar species.

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�?nbsp; 1产地样本数及无性系编号

Table 1.Sample number from provenances and clone No.

产地 Provenance	样本�?br/> Sample number	无性系编号 Clone No.
北京 Beijing	25	1 ~ 25
河北 Hebei	56	26 ~ 81
山东 Shandong	19	82�?3�?21�?22�?32�?34�?35�?37�?42 ~ 249�?51�?54�?55
河南 Henan	41	84 ~ 114�?23�?25 ~ 230�?57 ~ 259
山西 Shanxi	44	115 ~ 157�?60
陕西 Shaanxi	49	158 ~ 203�?38�?39�?61
甘肃 Gansu	5	204 ~ 208
安徽 Anhui	9	209 ~ 217
江苏 Jiangsu	4	218 ~ 220�?63

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�?nbsp; 2毛白杨核心种质排序表

Table 2.Rank of core germplasm ofP. tomentosa

顺序 Order	无性系编号 Clone No.	顺序 Order	无性系编号 Clone No.	顺序 Order	无性系编号 Clone No.	顺序 Order	无性系编号 Clone No.
1	156	18	265	35	227	52	161
2	258	19	263	36	264	53	113
3	236	20	233	37	211	54	160
4	253	21	225	38	196	55	259
5	266	22	267	39	208	56	203
6	262	23	218	40	176	57	158
7	261	24	222	41	195	58	110
8	231	25	270	42	138	59	155
9	34	26	197	43	193	60	272
10	241	27	256	44	152	61	202
11	271	28	269	45	184	62	154
12	242	29	220	46	180	63	255
13	252	30	250	47	181	64	151
14	186	31	191	48	89	65	201
15	240	32	219	49	26	66	47
16	237	33	72	50	178	67	147
17	268	34	215	51	126	68	109

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�?nbsp; 3不同取样比例种质总数及不同来源份�?/p>

Table 3.Total number of germplasms with different sampling ratios and the number of samples from different sources

比例 Ratio/%	北京 Beijing	河北 Hebei	山东 Shandong	河南 Henan	山西 Shanxi	陕西 Shaanxi	甘肃 Gansu	安徽 Anhui	江苏 Jiangsu	新疆 Xinjiang	杂种 Hybrid	总计 Total
100.0	25	56	19	41	44	49	5	9	4	1	19	272
25.0	0	4	4	8	8	18	1	2	4	1	18	68
20.0	0	3	3	6	4	14	1	2	4	1	17	55
15.0	0	2	3	3	1	7	1	2	4	1	17	41
10.0	0	1	3	2	1	3	0	0	2	1	14	27
5.0	0	1	1	1	1	2	0	0	0	0	8	14
2.5	0	0	0	1	1	1	0	0	0	0	4	7
2.0	0	0	0	1	1	0	0	0	0	0	3	5
1.5	0	0	0	1	1	0	0	0	0	0	2	4

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�?nbsp; 4不同取样比例微核心种质遗传多样性分枏�/p>

Table 4.Genetic diversity analysis of microcore germplasm with different sampling ratios

样本野�br/> Sample number (N)	取样比例 Sampling ratio/%	等位基因�?br/> Allele number (Na)	有效等位基因�?br/> Effective number of allele (Ne)	Shannon信息指数 Shannon information index (I)	观测杂合�?br/> Observed heterozygosity (Ho)	期望杂合�?br/> Expected heterozygosity (He)	平均多态信息指�?br/>Average polymorphic information index (PIC)
272	100.0	6.625	2.075	0.825	0.561	0.432	0.364
68	25.0	6.063	2.761	1.094	0.605	0.539	0.499
55	20.0	6.063	2.916	1.141	0.605	0.557	0.519
41	15.0	5.875	3.145	1.205	0.601	0.584	0.547
27	10.0	5.813	3.317	1.271	0.581	0.614	0.578
14	5.0	4.938	3.385	1.234	0.589	0.616	0.576
7	2.5	4.625	3.404	1.254	0.554	0.638	0.594
5	2.0	4.438	3.513	1.254	0.550	0.643	0.597
4	1.5	3.625	3.003	1.048	0.625	0.563	0.512

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�?nbsp; 5微核心种质（10%）与初始种质的遗传多样性比辂�/p>

Table 5.Comparison of genetic diversity between microcore germplasm (10%) and primary germplasm

群体 Population	N	Na	Ne	I	Ho	He	PIC
初始种质 Primary germplasm	272	6.625	2.075	0.825	0.561	0.432	0.364
微核心种� Microcore germplasm	27	5.813	3.317	1.271	0.581	0.614	0.578
保留� Retention rate/%	10	87.743	159.855	154.061	103.565	142.130	158.791
t	0.993 8
P	0.358 7 > 0.05

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�?nbsp; 6微核心种质（2%）与初始种质的遗传多样性比辂�/p>

Table 6.Comparison of genetic diversity between the microcore germplasm (2%) and primary germplasm

群体 Population	N	Na	Ne	I	Ho	He	PIC
初始种质 Primary germplasm	272	6.625	2.075	0.825	0.561	0.432	0.364
微核心种� Microcore germplasm	5	4.438	3.513	1.254	0.550	0.643	0.597
保留� Retention rate/%	2	66.989	169.301	152.000	98.039	148.843	164.011
t	0.999 4
P	0.356 2 > 0.05

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�?nbsp; 7微核心种质（15%）与初始种质的遗传多样性比辂�/p>

Table 7.Comparison of genetic diversity between the microcore germplasm (15%) and primary germplasm

群体 Population	N	Na	Ne	I	Ho	He	PIC
初始种质 Primary germplasm	272	6.625	2.075	0.825	0.561	0.432	0.364
微核心种� Microcore germplasm	41	5.875	3.145	1.205	0.601	0.584	0.547
保留� Retention rate/%	15	88.679	151.566	146.061	107.130	135.185	150.275
t	0.994 6
P	0.358 4 > 0.05

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�?nbsp; 8微核心种质（1.5%）与初始种质遗传多样性比辂�/p>

Table 8.Comparison of genetic diversity between the microcore germplasm (1.5%) and primary germplasm

群体 Population	N	Na	Ne	I	Ho	He	PIC
初始种质 Primary germplasm	272	6.625	2.075	0.825	0.561	0.432	0.364
微核心种� Microcore germplasm	4	3.625	3.003	1.048	0.625	0.563	0.512
保留� Retention rate/%	1.5	54.717	144.723	127.030	111.408	130.324	140.659
t	1.006 5
P	0.353 0 > 0.05

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�?nbsp; 916个SSR位点27份微核心种质的多态性分枏�/p>

Table 9.Polymorphism analysis of 27 accessions of microcore germplasm at 16 SSR locus

引物 Primer	位点 Locus	N	Na	Ne	I	Ho	He	PIC
1	Ptr_1_SSR2	27	5.000	3.291	1.300	0.593	0.696	0.640
2	Ptr_7_SSR14	27	7.000	4.500	1.649	0.815	0.778	0.744
3	Ptr_1_SSR1	27	3.000	1.573	0.655	0.333	0.364	0.327
4	Ptr_9_SSR3	27	4.000	2.282	1.040	0.222	0.562	0.512
5	Ptr_11_SSR1	27	7.000	4.084	1.595	0.556	0.755	0.718
6	Ptr_10_SSR1	27	4.000	2.809	1.136	0.852	0.644	0.574
7	Ptr_11_SSR8	27	5.000	1.421	0.653	0.148	0.296	0.283
8	Ptr_14_SSR12	27	7.000	4.166	1.636	0.852	0.760	0.726
9	Ptr_14_SSR7	27	5.000	3.827	1.465	0.852	0.739	0.698
10	Ptr_16_SSR3	27	6.000	1.369	0.651	0.111	0.270	0.263
11	Ptr_3_SSR13	27	7.000	3.044	1.364	0.259	0.671	0.615
12	Ptr_14_SSR11	27	6.000	2.553	1.239	0.778	0.608	0.566
13	Ptr_18_SSR17	27	10.000	6.568	2.032	0.815	0.848	0.830
14	Ptr_7_SSR15	27	9.000	6.688	2.015	0.926	0.850	0.833
15	Ptr_13_SSR6	27	5.000	3.539	1.401	0.889	0.717	0.672
16	Ptr_19_SSR1	27	3.000	1.353	0.503	0.296	0.261	0.241
平均� Mean		27	5.813	3.317	1.271	0.581	0.614	0.578

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�?nbsp; 10�?对引物构建的27份毛白杨微核心种质指纹图谰�/p>

Table 10.Fingerprint map of 27 microcore germplasms ofP. tomentosaconstructed using the top 8 pairs of primers bp

顺序 Order	无性系编号 Clone No.	引物 Primer
顺序 Order	无性系编号 Clone No.	1	2	3	4	5	6	7	8
1	156	378/378	393/396	185/185	369/372	271/281	241/244	332/332	333/345
2	258	382/390	363/366/399	185/187	372/372	269/283	238/241/244	332/332	337/355
3	236	374/382/390	360/363/399	185/185	369/369	269/283	238/241	332/332	335/337
4	253	378/378	360/396	185/185	372/372	271/287	238/241	294/294	333/337
5	266	390/390	357/363	183/183	269/269	275/275	238/238	330/330	333/333
6	262	374/378	360/396	185/185	372/372	271/283	241/244	332/332	333/333
7	261	382/390	363/396	185/187	269/269	269/269	238/241/250	332/332	337/337
8	231	376/378	393/396	185/185	369/372	271/271	241/244	332/332	333/345
9	34	382/390	363/366/399	185/187	372/372	269/283	238/241/244	332/332	337/355
10	241	378/378	393/396	185/185	372/372	271/271	238/244	332/332	333/353
11	271	374/382/390	360/363/399	185/185	369/369	269/269	238/241	332/332	333/337
12	242	382/390	363/366/399	185/187	372/372	269/283	238/241/244	332/332	337/355
13	252	378/378	396/396	185/185	369/372	271/281	241/244	332/332	333/345
14	186	382/390	363/396	185/187	369/369	269/269	238/241/250	332/332	335/337
15	240	378/378	393/396	185/185	372/372	271/271	238/244	332/332	333/353
16	237	382/390	363/366/399	185/187	372/372	269/283	238/241/244	332/332	337/355
17	268	378/378	360/396	185/185	369/372	271/271	241/244	332/332	333/345
18	265	378/382	360/399	185/185	372/372	283/287	238/241	294/332	333/337
19	263	382/390	360/363	185/185	372/372	269/281	238/250	332/338	333/337
20	233	378/378	396/396	185/185	366/369	271/271	241/241	332/332	331/333
21	225	382/390	363/366/399	185/187	372/372	269/283	238/241/244	332/332	337/355
22	267	390/390	363/363	185/187	269/269	285/285	238/238	330/334	333/333
23	218	378/378	360/360	185/185	366/366	271/271	241/241	332/332	331/353
24	222	382/390	363/366/399	185/187	372/372	269/283	238/241/244	332/332	337/355
25	270	378/378	396/396	185/185	369/372	271/271	241/244	332/332	333/345
26	197	382/390	360/363	185/185	372/372	269/281	238/250	332/332	333/337
27	256	378/382/390	360/363/399	185/185	372/372	271/283	238/241	294/332	335/337

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�?nbsp; 11�?对引物构建的27份毛白杨微核心种质指纹图谰�/p>

Table 11.Fingerprint map of 27 microcore germplasms ofP.tomentosa constructed using the last 8 pairs of primers bp

顺序 Order	无性系编号 Clone No.	引物 Primer
顺序 Order	无性系编号 Clone No.	9	10	11	12	13	14	15	16
1	156	253/262	356/356	373/373	327/348	177/179	347/355	339/345	289/292
2	258	250/259	356/356	353/373	327/336	175/179	345/361/363	339/342/348	289/289
3	236	259/259	356/356	353/353	327/327	171/181	355/361/363	342/345	280/289
4	253	253/259	356/356	373/373	327/336	173/177	339/365	339/339	289/289
5	266	259/259	340/340	371/371	321/330	193/203	363/363	336/342	289/289
6	262	262/262	356/356	353/353	327/327	179/181	339/365	336/345	280/289
7	261	256/259	356/356	353/353	327/336	171/173/175	359/363	339/342	280/289
8	231	253/262	356/356	373/373	327/348	177/181	347/355	339/345	289/292
9	34	250/259	356/356	353/373	327/336	175/179	345/361/363	339/348	289/289
10	241	253/259	356/356	375/375	306/327	173/173	347/373	336/342	289/289
11	271	256/262	356/356	353/353	327/327	171/171	355/361/363	342/345	280/289
12	242	250/259	356/356	353/373	327/336	175/179	345/361/363	339/342/348	289/289
13	252	253/262	356/356	373/373	327/348	177/179	347/355	339/345	289/289
14	186	256/259	356/356	353/353	327/336	171/173/175	359/363	339/342	280/289
15	240	253/259	356/356	375/375	306/327	173/173	347/373	336/342	289/289
16	237	250/259	356/356	353/373	327/336	175/179	345/361/363	339/342/348	289/289
17	268	253/262	356/356	373/373	327/348	177/177	347/355	342/345	280/289
18	265	259/262	356/356	387/387	327/327	173/179	361/365	336/339	289/289
19	263	256/262	356/356	353/353	327/336	173/191	361/363	339/342	289/289
20	233	259/262	326/334	373/373	327/327	179/181	347/355	339/345	289/289
21	225	250/259	356/356	353/373	327/336	175/179	345/361/363	339/342/348	289/289
22	267	259/262	316/328	369/369	321/330	201/201	363/365	342/348	289/289
23	218	259/259	326/334	353/377	327/327	177/179	355/355	339/339	289/289
24	222	250/259	356/356	353/373	327/336	175/179	345/361/363	339/342/348	289/289
25	270	253/262	356/356	373/373	327/348	177/179	347/355	339/345	289/289
26	197	256/262	356/356	353/353	327/336	173/191	361/363	339/342	289/289
27	256	259/262	356/356	353/353	327/336	171/173	361/363/365	339/339	289/289

下载: 导出CSV

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