基于EST-SSR标记鉴定猴耳环自由授粉的全同胞子代

doi:10.12302/j.issn.1000-2006.202107029

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南京林业大学学报（自然科学版） ›› 2022, Vol. 46 ›› Issue (4): 95-101.doi: 10.12302/j.issn.1000-2006.202107029

基于EST-SSR标记鉴定猴耳环自由授粉的全同胞子代

李丹丹¹^,²^,³(), 翁启杰¹, 甘四明¹^,³, 周长品¹, 黄世能¹, 李梅¹^,^*()

1.中国林业科学研究院热带林业研究所, 热带林业研究国家林业和草原局重点实验室,广东广州 510520
2.南京林业大学林学院, 江苏南京 210037
3.中国林业科学研究院,林木遗传育种国家重点实验室, 北京 100091

收稿日期:2021-07-20 修回日期:2021-11-29 出版日期:2022-07-30 发布日期:2022-08-01
通讯作者: 李梅
基金资助:
中央级公益性科研院所基本科研业务费专项资金项目(CAFYBB2018SY019);广东省林业科技创新项目(2017KJCX004)

Identification of full-sib seedlings from an open-pollinated family of Archidendron clypearia based on EST-SSR markers

LI Dandan¹^,²^,³(), WENG Qijie¹, GAN Siming¹^,³, ZHOU Changpin¹, HUANG Shineng¹, LI Mei¹^,^*()

1. Key Laboratory of National Forestry and Grassland Administration on Tropical Forestry Research, Research Institute of Tropical Forestry, Chinese Academy of Forestry, Guangzhou 510520, China
2. College of Forestry, Nanjing Forestry University, Nanjing 210037, China
3. State Key Laboratory of Tree Genetics and Breeding, Chinese Academy of Forestry, Beijing 100091, China

Received:2021-07-20 Revised:2021-11-29 Online:2022-07-30 Published:2022-08-01
Contact: LI Mei

摘要/Abstract

摘要：

【目的】猴耳环是重要的药用树种,具有较好的工业应用价值。利用EST-SSR标记对猴耳环自然群体1株母树的自由授粉子代进行父本鉴定,从而确定全同胞子代,为基于全同胞家系的后续研究提供材料基础。【方法】以自然群体中母树ELS31自由授粉产生的1 489株子代及该群体内挂果的38株候选父本为材料,利用15个EST-SSR标记检测子代多样性和标记多态性,基于最大似然法鉴定各子代的父本,母本父本均相同的子代即构成全同胞家系,并估算群体内花粉传播距离,检验各父本对应的全同胞家系的多样性,通过卡方检验判断标记是否合乎预期的孟德尔分离比。【结果】15个EST-SSR标记的引物(对)共扩增出89个等位片段。子代群体期望杂合度(H_e)为0.525,表明群体多样性为中等水平;基于自然群体18株无亲缘关系的单株估算的标记平均多态性信息量(PIC)为0.736,表明标记多态性高。在1 489株自由授粉的子代中,确定了857株子代(57.6%)的34株父本。子代数最多的10个父本产生的子代数为26~184株,其他24个父本仅共产生子代139株。未发现自交子代,表明猴耳环是异交物种,自交的可能性极低。对子代数量20株以上的10个父本对应的全同胞家系观测杂合度(H_o)为0.502~0.693,H_e为0.417~0.544,各家系均是H_o大于H_e,表明存在一定程度的杂合子过剩。花粉传播的范围为10.0~559.1 m,平均119.2 m,但主要传播距离在150 m以内。716株子代(83.5%)的父本(10株)与母树距离在150 m以内。距离最远的父本ELS01 (559.1 m)和ELS02 (552.2 m)分别仅产生了9和12株子代。15个标记在子代20株以上的部分或全部全同胞家系中均有不同程度的偏分离,平均每家系的偏分离标记数为8.6个;偏分离最严重的是标记ARCeSSR141,在父本ELS30的全同胞家系中卡方(χ²)值为164.55。【结论】基于15个EST-SSR标记鉴定了猴耳环自然群体1株母本的1 489株自由授粉子代的父本,获得了子代20株以上的10个父本的全同胞家系。这为后续遗传测定、遗传图谱构建和数量性状位点定位等研究提供了材料基础。

关键词: 猴耳环, EST-SSR标记, 全同胞家系, 子代多样性, 父本分析

Abstract:

【Objective】 Archidendron clypearia is an important medicinal tree species which occurs widely in tropical Asian regions and has valuable resources for industrial applications. This study aimed to identify paternal parents in an open-pollinated family in a natural forest of A. clypearia, based on the expressed sequence tag derived simple sequence repeats (EST-SSR) markers, to determine which are full-sib seedlings, and therefore provide full-sib families for subsequent studies. 【Method】 The maternal parent ELS31 and 38 fruited candidate paternal parents from a natural population, together with 1 489 open-pollinated seedlings of ELS31, were studied using 15 EST-SSR markers. Seedling diversity and marker polymorphism were estimated. Paternal parents of the seedlings were identified using the method of logarithm of odds, and full-sibs originating from the same maternal and paternal parents were identified. The pollen dispersal range was determined from the distance between the maternal and paternal parents. The diversity of father-derived full-sib families was also examined. A Chi-square test was carried out for the markers to determine whether to follow the Mendelian expectation of segregation ratios. 【Result】 Primer pairs of the 15 EST-SSR markers amplified a total of 89 alleles across the seedling population. The expected heterozygosity (H_e) was 0.525 for the seedling population, indicating the moderate genetic diversity. The polymorphism information content (PIC) estimated with 18 unrelated trees from the natural forest was averaged at 0.736 over the 15 EST-SSRs, suggesting a high level of marker polymorphism. Of the 1 489 open-pollinated sibs, 857 (57.6%) originated from 34 definite paternal parents, with the top ten paternal parents each contributing to 26-184 sib seedlings and other 24 paternal parents contributing to 139 sib seedlings. Selfed sibs were not found, indicating that A. clypearia is an outcrossing species with an extremely low chance of selfing. For the top ten full-sib families, H_o ranged from 0.502 to 0.693, and H_e ranged from 0.417 to 0.544. Higher H_o estimates in these families indicate a certain magnitude of heterozygote excess. The pollen dispersal distance ranged from 10.0 to 559.1 m, with a mean of 119.2 m and the most effective range being within 150 m. A subset of 716 seedlings (83.5%) were generated from the 10 paternal parents within the range of 150 m from the maternal parent. The most distant paternal parents ELS01 (559.1 m) and ELS02 (552.2 m) contributed to 9 and 12 seedlings, respectively. The 15 EST-SSRs distorted more or less from the Mendelian expectation in segregation among part or all of the top ten full-sib families. The mean number of the distorted loci was 8.6 in the top ten full-sib families. The most distorted locus was ARCeSSR141 in the ELS30 fathered family, with a χ² value of 164.55.【Conclusion】 Paternal parents were identified for an open-pollinated family (1 489 seedlings) of A. clypearia using EST-SSR markers, resulting in 10 full-sib families with each comprising more than 20 seedlings. The full-sib families identified here will provide a useful plant material for further studies such as genetic testing, genetic map construction and quantitative trait locus detection.

Key words: Archidendron clypearia, EST-SSR marker, full-sib family, diversity of sibs, paternity analysis

中图分类号:

S722

李丹丹,翁启杰,甘四明,等. 基于EST-SSR标记鉴定猴耳环自由授粉的全同胞子代[J]. 南京林业大学学报（自然科学版）, 2022, 46(4): 95-101.

LI Dandan, WENG Qijie, GAN Siming, ZHOU Changpin, HUANG Shineng, LI Mei. Identification of full-sib seedlings from an open-pollinated family of Archidendron clypearia based on EST-SSR markers[J].Journal of Nanjing Forestry University (Natural Science Edition), 2022, 46(4): 95-101.DOI: 10.12302/j.issn.1000-2006.202107029.

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序号 No.	EST-SSR标记 EST-SSR marker	等位片段范围/bp ASR	N_a	N_e	H_o	H_e	多态性信息量 PIC
1	ARCeSSR006	190~196	4	1.220	0.191	0.185	0.622
2	ARCeSSR075	156~162	4	1.991	0.692	0.498	0.712
3	ARCeSSR095	125~149	12	2.848	0.657	0.649	0.885
4	ARCeSSR141	167~175	5	3.318	0.823	0.699	0.807
5	ARCeSSR266	143~149	4	2.214	0.733	0.529	0.758
6	ARCeSSR277	195~201	4	1.811	0.554	0.448	0.753
7	ARCeSSR288	106~118	4	1.780	0.629	0.438	0.577
8	ARCeSSR304	125~149	7	2.047	0.742	0.512	0.859
9	ARCeSSR366	166~181	6	3.037	0.667	0.671	0.762
10	ARCeSSR425	127~145	10	2.235	0.523	0.553	0.812
11	ARCeSSR448	288~297	4	1.908	0.445	0.476	0.681
12	ARCeSSR464	197~207	6	1.983	0.431	0.496	0.619
13	ARCeSSR474	114~135	6	2.225	0.482	0.551	0.749
14	ARCeSSR649	282~288	4	2.084	0.470	0.520	0.624
15	ARCeSSR665	177~199	9	2.856	0.720	0.650	0.826
平均值±标准差mean±SD			5.9±2.5	2.237±0.5	0.584±0.158	0.525±0.120	0.736±0.091

序号 No.	父本 paternal parent	子代数量(占比/%) number of sibs(proportion)	亲本距离/m parental distance	H_o	H_e	父本概率对数值 LOD
1	ELS30	184 (21.5)	50.0	0.584	0.484	0.015~8.608
2	ELS32	139 (16.2)	10.0	0.502	0.417	0.034~6.440
3	ELS35	122 (14.2)	100.0	0.693	0.530	0.229~19.952
4	ELS24	55 (6.4)	111.8	0.595	0.485	0.003~14.193
5	ELS33	53 (6.2)	35.0	0.648	0.517	0.121~10.488
6	ELS25	43 (5.0)	90.1	0.693	0.544	0.213~15.801
7	ELS34	36 (4.2)	50.0	0.646	0.518	0.008~14.376
8	ELS14	31 (3.6)	373.0	0.533	0.447	0.004~4.853
9	ELS29	29 (3.4)	55.9	0.561	0.445	1.243~16.487
10	ELS27	26 (3.0)	78.1	0.635	0.503	0.148~14.826
其他others	24株	139 (16.2)	70.7~559.1	0.467~0.757	0.233~0.539	0.008~15.826
合计total	34株	857

序号 No.	标记 marker	ELS30	ELS32	ELS35	ELS24	ELS33	ELS25	ELS34	ELS14	ELS29	ELS27
1	ARCeSSR141	164.6^***	64.9^***	110.3^***	55.0^***	28.7^***	28.5^***	28.4^***	9.4^**	25.2^***	7.5^**
2	ARCeSSR266	40.2^***	62.2^***	117.0^***	—	25.8^***	17.0^***	7.1^***	0.8	—	7.5^***
3	ARCeSSR277	5.3^**	0.3	—	46.3^***	—	19.6^***	36.0^***	3.9	—	2.0
4	ARCeSSR304	67.1^***	44.7^***	122.0^***	2.8	18.3^***	15.3^***	9.1^**	17.2^***	20.6^**	3.2
5	ARCeSSR665	68.4^***	6.4^*	68.7^***	18.2^***	26.4^***	38.1^***	12.7^***	12.9^***	5.1	26.0^***
6	ARCeSSR425	55.4^***	9.4^**	1.0	1.0	9.0^**	11.9^***	5.4^**	1.2	2.3	7.5^**
7	ARCeSSR464	3.9	4.5^**	31.6^***	0.7	7.1^*	4.00	7.1^***	1.7	2.3	0.7
8	ARCeSSR006	—	-	—	-	—	5.2^**	1.0	—	2.8	-
序号 No.	标记 marker	ELS30	ELS32	ELS35	ELS24	ELS33	ELS25	ELS34	ELS14	ELS29	ELS27
9	ARCeSSR095	43.3^***	3.0	51.3^***	20.6^***	4.2	21.8^***	4.2	4.4	5.4	22.2^***
10	ARCeSSR075	151.4^***	—	117.0^***	17.3^***	20.5^***	12.3^***	21.8^***	3.3	0.3	3.9
11	ARCeSSR649	5.9	21.4^***	0.0	8.8^**	7.9^*	0.6	8.0^**	1.1	1.3	1.4
12	ARCeSSR288	56.6^***	0.6	44.9^***	19.0^***	15.1^***	16.4^***	0.4	12.5^***	4.2	4.9
13	ARCeSSR366	102.5^***	51.0^***	36.8^***	18.3^***	41.7^***	33.6^***	11.3^***	8.9^*	0.3	14.4^***
14	ARCeSSR448	0.2	0.0	1.6	2.6	6.3	0.1	12.2^***	0.4	4.5	1.0
15	ARCeSSR474	21.6^***	14.2^***	39.5^***	6.6^**	5.0^*	14.4^***	3.7	0.3	4.9^*	0.2
	偏分离标记数 No. loci distorted	11	9	10	9	11	12	11	5	3	6

基于EST-SSR标记鉴定猴耳环自由授粉的全同胞子代

Identification of full-sib seedlings from an open-pollinated family of Archidendron clypearia based on EST-SSR markers

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