90份鳄梨种质资源AFLP遗传多样性分析

董美超; 杨帆; 李进学; 付小猛; 高俊燕; 周东果; 王绍华; 龙春瑞; 郭莉娜; 岳建强

doi:10.19303/j.issn.1008-0384.2020.01.003

90份鳄梨种质资源AFLP遗传多样性分析

doi: 10.19303/j.issn.1008-0384.2020.01.003

云南省农业科学院热带亚热带经济作物研究所，云南　保山　678000

基金项目: 云南省农业基础研究联合专项[2017FG001（-060）]

详细信息

作者简介:
董美超（1985−），女，硕士，助理研究员，研究方向：热带果树育种（E-mail：bluetide14@126.com）

通讯作者:
岳建强（1967−），男，硕士，研究员，研究方向：果树育种（E-mail：yjq7009@163.com）

中图分类号: S 667.9
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- 被引次数: 0
出版历程
- 收稿日期: 2019-09-06
- 修回日期: 2019-11-25
- 刊出日期: 2020-01-01

AFLP Molecular Markers-based Genetic Diversity Analysis on 90 Avocado Germplasms

Tropical and Subtropical Economic Crops Research Institute, Yunnan Academy of Agricultural Sciences, Baoshan, Yunnan　678025, China

摘要

摘要: 目的本研究对采自中国云南德宏、保山、西双版纳、普洱、红河和缅甸克钦邦、掸邦地区共90份鳄梨种资资源进行遗传多样性分析，为其新品种选育和种质创新提供参考依据。方法采用CTAB法提取鳄梨叶片基因组DNA，利用扩增片段长度多态性分子标记技术，对90份种质资源的基因组DNA进行酶切、连接、预扩增、选择性扩增，电泳分离，银染显色。电泳结果得到“0，1”矩阵，使用POPGENE 32软件计算每对引物的多态性条带、多态性比率、有效等位基因数、遗传多样性指数等指标，同时使用NTSYSpc-2.11F计算种质间遗传相似系数，根据相似性系数进行UPGMA聚类分析和PCA主效应分析，对鳄梨种质资源进行分类。结果从24对AFLP引物组合中，筛选出8对扩增条带清晰、多态性高的引物组合，8对引物共扩增出1 165个条带，其中多态性条带有1 163个，多态位点百分率为99.83%；有效等位基因数（Ne）平均1.294 4个；Nei’s基因多样性指数（H）平均0.209 5；Shannon信息指数（I）平均0.353 0。根据遗传相似系数进行聚类分析，在遗传相似系数0.752处可划分为4个类群，第I类群有1份保山种质70号；第II类群有24份种质；第III类群有1份西双版纳种质59号；第IV类群有64份种质。在遗传相似系数0.763处可将第IV类群划分为3个亚群（A、B和C）。用PCA法对90份鳄梨种质AFLP标记结果进行主效应分析，显示了不同种质的分类位置，主效应分析结果与分子聚类结果基本一致，呈一定的地域性分布规律。结论 90份种质资源的遗传多样性较为丰富，59号和70号相对特殊，在种质创新中应给予重点关注。
- 鳄梨 /
- AFLP /
- 遗传多样性
Abstract: Objective Genetic relationship of 90 avocado (Persea americana Mill.) germplasms collected from Dehong, Baoshan, Xishuangbanna, Puer, and Honghe in Yunnan, China and Shan and Kachin States of Burma was analyzed based on AFLP molecular markers for breeding reference and resource information. Method Genomic DNAs were extracted from the leaves of the avocado plants following the CTAB method. They were amplified using the sequence-related amplified polymorphism molecular markers to determine their genetic diversity and similarity. Separation of the amplified fragments was performed on 5% denaturing polyacrylamide gels stained with AgNO₃ to obtain the “0,1” matrix. The number of polymorphic loci, percentage of polymorphic loci, effective number of alleles, and indices of genetic diversity were estimated by POPGENE version 32. The genetic similarity estimated by NTSYSpc-2.11F was used for UPGMA (unweighted pair group method analysis) and PCA (principal component analysis) to classify the avocado germplasms. Result Eight primers with strong polymorphism and high repeatability were screened from 24 AFLP primers generating a total of 1 165 bands. Of the bands, 1 163 (99.83%) showed polymorphism. On average, the number of effective alleles was 1.294 4; Nei's gene diversity (H), 0.209 5; and Shannon's information index (I), 0.353 0. At the genetic similarity index (GS) of 0.752, the germplasms were classified into 4 groups. A single avocado germplasm, No. 70, collected from Baoshan was placed in Group I. Group II had 22 germplasms; Group III, one germplasm, No. 59, from Xishuangbanna; and Group IV, the 64 remainders. The germplasms in Group IV were further divided into Subgroups A, B and C at GS of 0.763. A similar grouping result was obtained from the cluster analysis on the PCR data applied AFLP markers confirming these germplasms shared a high similarity within a same region. Conclusion The 90 collected avocado germplasms were found to be relatively high in genetic diversity. Among them, No. 59 and 70 stood out as two most unique varieties.
- Avocado (Persea americana Mill) /
- AFLP /
- genetic diversity

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图 1 引物E-AGC/ M-CAC对60～90号鳄梨种质的AFLP扩增结果

Figure 1. PCR amplification patterns by AFLP primer E-AGC/M-CAC of germplasms No.60–90

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图 2 90份鳄梨种质材料的AFLP聚类分析

Figure 2. Dendrogram of 90 accessions of avocado germplasms with AFLP markers

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图 3 鳄梨种质AFLP主效应分析

Figure 3. Principal components analysis on avocado germplasms using AFLP data

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表 1 供试鳄梨种质资源信息

Table 1. Avocado germplasms studied

序号 Number	种质编号 Germplasm code	序号 Number	种质编号 Germplasm code	序号 Number	种质编号 Germplasm code
来源地：缅甸克钦邦八莫市 Origin: Bhamo, Kachin state, Myanmar		来源地：云南省普洱市孟连县 Origin: Menglian County, Pu'er City, Yunnan Province		62	EL0062
				63	EL0063
				64	EL0064
1	EL0001	32	EL0032	65	EL0065
2	EL0002	33	EL0033	来源地：云南省德保山市潞江坝 Origin: Lujiangba, Baoshan City, Yunnan Province
3	EL0003	34	EL0034
4	EL0004	35	EL0035
5	EL0005	36	EL0036	66	EL0066
6	EL0006	37	EL0037	67	EL0067
7	EL0007	38	EL0038	68	EL0068
8	EL0008	39	EL0039	69	EL0069
9	EL0009	40	EL0040	70	EL0070
10	EL0010	41	EL0041	来源地：云南省德宏州盈江县 Origin: Yingjiang County, Dehong Prefecture, Yunnan Province
11	EL0011	42	EL0042
12	EL0012	43	EL0043
13	EL0013			71	EL0071
来源地：缅甸掸邦南坎 Origin: Namhkam, Shan State, Myanmar		来源地：云南省热带作物科学研究所 Origin: Yunnan Institute of Tropical Crops		72	EL0072
				73	EL0073
				74	EL0074
14	EL0014	44	EL0044	75	EL0075
15	EL0015	45	EL0045	76	EL0076
16	EL0016	46	EL0046	来源地：云南省德宏州瑞丽市 Origin: Ruili City, Dehong Prefecture, Yunnan Province
17	EL0017	47	EL0047
18	EL0018	48	EL0048
19	EL0019	49	EL0049	77	EL0077
20	EL0020	50	EL0050	78	EL0078
21	EL0021	51	EL0051	79	EL0079
22	EL0022	52	EL0052	80	EL0080
23	EL0023	53	EL0053	81	EL0081
来源地：云南省红河州元阳县 Origin: Yuanyang County, Honghe Prefecture, Yunnan Province		54	EL0054	82	EL0082
		55	EL0055	83	EL0083
		56	EL0056	84	EL0084
24	EL0024	57	EL0057	85	EL0085
25	EL0025	来源地：云南省西双版纳州景洪县 Origin: Jinghong County, Xishuangbanna Prefecture, Yunnan Province		86	EL0086
26	EL0026			来源地：云南省德宏州芒市 Origin: Mangshi City, Dehong Prefecture, Yunnan Province
27	EL0027
28	EL0028	58	EL0058	87	EL0087
29	EL0029	59	EL0059	88	EL0088
30	EL0030	60	EL0060	89	EL0089
31	EL0031	61	EL0061	90	EL0090

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表 2 引物系列

Table 2. Primer sequence

接头与引物 Connector and primer	系列（5′-3′） Sequence
Eco RI 接头1（Eco RI Connector 1）	CTCGTAGACTGCGTACC
Eco RI 接头2 （Eco RI Connector 2）	AATTGGTACGCAGTCTAC
Mse I 接头1 （Mse IConnector 1）	GACGATGAGTCCTGAG
Mse I 接头2 （Mse I Connector 2）	TACTCAGGACTCAT
3-2引物（3-2 primer）	E-AAC/M-CAC
3-3引物（3-3 primer）	AAC/M-CAG
3-6引物（3-6 primer）	E-AAC/M-CTC
4-2引物（4-2 primer）	E-AAG/M-CAC
7-1引物（7-1 primer）	E-ACC/M-CAA
7-3引物（7-3 primer）	E-ACC/M-CAG
9-2引物（9-2 primer）	E-AGC/M-CAC
10-2引物（10-2 primer）	E-AGG/M-CAC

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表 3 8对引物组合扩增的AFLP条带

Table 3. Amplification bands with 8 pairs of AFLP primers

引物组合 Primer pair	总条带数 Total bands	多态性条带数 Polymorphic bands	多态性比率 Polymorphic rate/%	有效等位基因数 Ne	Nei’s基因多样性 H	Shannon’s多样性 I
E-AAC/M-CAC	144	144	100	1.306 1	0.215 3	0.359 9
F-AAC/M-CAG	145	144	99.31	1.257 9	0.188 5	0.325 0
E-AAC/M-CTC	136	135	99.26	1.329 1	0.225 6	0.372 2
E-AAG/M-CAC	147	147	100	1.294 8	0.209 9	0.353 3
E-ACC/M-CAA	147	147	100	1.322 5	0.225 8	0.374 3
E-ACC/M-CAG	150	150	100	1.268 6	0.197 2	0.337 9
E-AGC/M-CAC	141	141	100	1.313 7	0.218 8	0.365 2
E-AGG/M-CAC	155	155	100	1.262 1	0.194 9	0.335 8
平均 Mean	145.63	145.38	99.83	1.294 4	0.209 5	0.353 0
总和 Total	1 165	1 163

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