Genetic structure of different natural <i>Osmanthus fragrans</i> populations based on AFLP method

XU Yichun; HU Shaoqing; ZHAO Hongbo

doi:10.11833/j.issn.2095-0756.2014.02.009

Volume 31 Issue 2

Mar. 2014

Turn off MathJax

Article Contents

Article Navigation > Journal of Zhejiang A&F University > 2014 > 31(2): 217-223

XU Yichun, HU Shaoqing, ZHAO Hongbo. Genetic structure of different natural Osmanthus fragrans populations based on AFLP method[J]. Journal of Zhejiang A&F University, 2014, 31(2): 217-223. DOI: 10.11833/j.issn.2095-0756.2014.02.009

Citation:

XU Yichun, HU Shaoqing, ZHAO Hongbo. Genetic structure of different natural Osmanthus fragrans populations based on AFLP method[J]. Journal of Zhejiang A&F University, 2014, 31(2): 217-223. DOI: 10.11833/j.issn.2095-0756.2014.02.009

Genetic structure of different natural Osmanthus fragrans populations based on AFLP method

DOI: 10.11833/j.issn.2095-0756.2014.02.009

1.
The Nurturing Station for the State Key Laboratory of Subtropical Silvicuture, Zhejiang A & F University, Lin'an 311300, Zhejiang, China
2.
College of Civil Engineering and Architecture, Zhejiang Sci-Tech University, Hangzhou 310018, Zhejiang, China
3.
School of Landscape Architecture, Zhejiang A & F University, 311300 Lin'an, Zhejiang, China

Received Date: 2013-09-01
Rev Recd Date: 2013-10-29
Publish Date: 2014-04-20

Abstract

Osmanthus fragrans is being concerned in recent years due to its high economic value and ornamental value. It can provide significant evidences for breeding of new cultivars and protecting of natural populations with studying on the genetic diversity of natural populations of O. fragrans. The genetic diversity of 96 O. fragrans individuals of two natural populations from Quannan County of Jiangxi Province and Changting County of Fujian Province was estimated using amplified fragment length polymorphism (AFLP) method. A total number of 330 reproducible bands were amplified using seven AFLP primer combinations, and 276 bands were polymorphic with a proportion of 83.64%. At the species level, Shannon's information index (I) was 0.428 3, the Nei's genetic diversity (H_e) was 0.285 6, which indicated the genetic diversity of O. fragrans was rich; at the population level, Shannon's information index and Nei's genetic diversity of Fujian population was higher 1.5 than Jiangxi population, that showed Fujian population owned much rich genetic resource. Moreover, AMOVA indicated the most total genetic variation was within populations with a proportion of 71% and the less was among the populations with a proportion of 29%. Meanwhile, a certain extent level of genetic differentiation (G_st=0.161 6) was detected among the populations and genetic flow was 2.594 9.
- botany,
- Osmanthus fragrans,
- AFLP,
- genetic diversity,
- genetic structure,

Relative Article

[1]	DENG Lili, LIU Qinghua, ZHOU Zhichun, GAO Kai, LUO Dinghui. Genetic diversity analysis and core collection of pinewood nematodiasis-resistant Pinus massoniana germplasm resources . Journal of Zhejiang A&F University, 2024, 41(1): 67-78. doi: 10.11833/j.issn.2095-0756.20230333
[2]	HUANG Jinchun, WAN Siqi, CHEN Yang, LI Lihong, ZHANG Zili, ZHU Jianjun, WU Mei, XING Bingcong, SHAO Qingsong, LU Chenfei. Genetic diversity of Anoectochilus roxburghii based on ISSR and SRAP molecular markers . Journal of Zhejiang A&F University, 2023, 40(1): 22-29. doi: 10.11833/j.issn.2095-0756.20220473
[3]	LI Yaping, DAI Huiming, JIANG Wu, CHEN Jiadong, DUAN Xiaojing, TAO Zhengming. Genetic diversity of Polygonatum spp. from different production areas based on SRAP markers . Journal of Zhejiang A&F University, 2023, 40(3): 658-664. doi: 10.11833/j.issn.2095-0756.20220436
[4]	WANG Jie, ZHU Xipeng, WANG Tengfei, ZHU Jianjun, LI Wenjun, XING Bingcong, ZHENG Ying. Genetic diversity analysis of Bletilla striata germplasm by ISSR and SRAP markers . Journal of Zhejiang A&F University, 2023, 40(2): 321-329. doi: 10.11833/j.issn.2095-0756.20210690
[5]	ZHENG Liuhui, ZHAN Liyun, HOU Yu, YU Weiwu, ZENG Yanru, DAI Wensheng. SSR analysis of genetic diversity of male Torreya grandis . Journal of Zhejiang A&F University, 2022, 39(2): 329-337. doi: 10.11833/j.issn.2095-0756.20210279
[6]	WANG Li, XU Hao, YU Yundong, ZHANG Haizhen, GAO Yanhui, SHEN Xiao, JIN Chenying, CHEN Yimin. Genetic diversity analysis of Hippeastrum rutilum cultivars based on SCoT markers . Journal of Zhejiang A&F University, 2020, 37(5): 930-938. doi: 10.11833/j.issn.2095-0756.20190614
[7]	WANG Xiaodong, WANG Junyu, ZHOU Huanhuan, FU Lucheng, WANG Bin, ZHANG Rumin, GAO Yan. Effects of simulated acid rain and heat stress on antioxidant enzyme activities and non-structural carbon metabolism of Osmanthus fragrans 'Hangzhou Huang' . Journal of Zhejiang A&F University, 2019, 36(1): 54-61. doi: 10.11833/j.issn.2095-0756.2019.01.008
[8]	WANG Luoyun, HE Caiyun, LUO Hongmei, ZHANG Jianguo, DUAN Aiguo, ZENG Yanfei. Genetic variation of 7 subspecies and 26 important cultivars in Hippophae rhamnoides . Journal of Zhejiang A&F University, 2019, 36(4): 670-677. doi: 10.11833/j.issn.2095-0756.2019.04.006
[9]	CHAI Xiaojuan, GUO Weilong, CHEN Hui, WANG Junrong, XIE Dezhi, JIN Shuihu. Genetic diversity of Juncus effusus germplasm by ISSR markers . Journal of Zhejiang A&F University, 2017, 34(3): 552-558. doi: 10.11833/j.issn.2095-0756.2017.03.022
[10]	LIU Yuejun, ZHANG Yuan, JIANG Yanfeng, LIU Jingjing. Genetic diversity of Polygonatum germplasm . Journal of Zhejiang A&F University, 2016, 33(6): 1085-1091. doi: 10.11833/j.issn.2095-0756.2016.06.023
[11]	WU Yueyan, TAO Qiaojing, LI Bo, XU Danye. An optimal SRAP-PCR system of Rhododendron hybridum and its genetic diversity analysis with SRAP marker . Journal of Zhejiang A&F University, 2013, 30(6): 844-851. doi: 10.11833/j.issn.2095-0756.2013.06.007
[12]	ZHANG Zhen, ZHANG Han-guo, ZHANG Lei, ZHU Hang-yong, LI Xue-feng. Diversity analysis with RAPD for natural and breeding populations of Larix gmelinii . Journal of Zhejiang A&F University, 2012, 29(1): 130-136. doi: 10.11833/j.issn.2095-0756.2012.01.022
[13]	GAO Yan-hui, FAN Min-liang, LUO Wen-jian, HUANG Hua-hong, TONG Zai-kang. RAPD analysis of genetic diversity for Pseudolarix amabilis： a critically endangered plant . Journal of Zhejiang A&F University, 2011, 28(5): 815-822. doi: 10.11833/j.issn.2095-0756.2011.05.023
[14]	FAN Fan-rong. Genetic diversity of endangered Artocarpus hypargyreus . Journal of Zhejiang A&F University, 2010, 27(2): 266-271. doi: 10.11833/j.issn.2095-0756.2010.02.017
[15]	LI Jie, XIONG Zhi, ZHANG Cheng-gang. Genetic diversity of Alnus nepalensis in Yunnan revealed by RAPD-PCR . Journal of Zhejiang A&F University, 2008, 25(1): 16-21.
[16]	CAO Fu-liang, HUA Zhe-bin, WANG Gui-bin, ZHANG Wang-xiang. Genetic diversity in wild populations of Ginkgo biloba using random amplified polymorphic DNA (RAPD) analysis . Journal of Zhejiang A&F University, 2008, 25(1): 22-27.
[17]	WANG Zu-liang, DING Li-xia, ZHAO Ming-shui, CHENG Xiao-yuan, SHEN Qian. Genetic diversity of Ostrya rehderiana revealed by RAPD markers . Journal of Zhejiang A&F University, 2008, 25(3): 304-308.
[18]	CHANG Bing-hua, HUYong-hong, XUYe-gen, ZHANG Qiu-xing, ZHANG Wan-li. Ultrastructures of petal surface of Osmanthus fragrans cultivars . Journal of Zhejiang A&F University, 2007, 24(5): 533-537.
[19]	HU Shao-qing, XUAN Zi-can, ZHOU Xu-lang, WU Guang-hong. Taxon and clear of Osmanthus fragrans cultivars in Hangzhou . Journal of Zhejiang A&F University, 2006, 23(2): 179-187.
[20]	TANG Juan-juan, FAN Yi-rong, ZHU Mu-yuan. Analysis of the genetic diversity of Pinus taiwanensis populations . Journal of Zhejiang A&F University, 2003, 20(1): 23-26.

References

[1]	WANG Xianrong,YUAN Fayin,CHEN Xin,et al. Quantitative characteristics of species in wild community of Osmanthus fragrans in Guihuaxia,Liuyang,Hunan[J]. J Nanjing For Univ Nat Sci Ed,2004,28(supp):41-44.
[2]	WANG De'e,WANG Zonghai. Study on breeding and wild population of Osmanthus fragrans in south slopes of Lushan[J]. For Sci Technol,1997(10):18-21.
[3]	LEI Ruihu,YU Xunlin,WANG Liqing. Research on the introduction of Osmanthus fragrans community in China[J]. J Anhui Agric Sci,2008,36(6):2337,2354.
[4]	DONG Jianwen,FAN Xiaoming,WU Donglai,et al. Quantitative characteristics of species in secondary forest community of Osmanthus fragrans in Shifengzhai of Changting County,Fujian Province[J]. J Plant Resour Environ,2002,11(4):40-44.
[5]	HAO Riming,ZHAO Hongbo,WANG Jinhu,et al. Observation and study on breeding system of wild Osmanthus fragrans[J]. J Plant Resour Environ,2011,20(1):17-24.
[6]	DUAN Yifan,TAN Zhiming,LIU Yuanbiao,et al. Resources investigation of ancient tree and wild tree of Osmanthus fragrans in Quannan County of Jiangxi Province[J]. J Plant Resour Environ,2012,21(2):118-120.
[7]	YEH F C,YANG R C,BOYLE T B J,et al. PopGene32,Microsoft Windows-Based Freeware for Population Genetic Analysis Version 1.32[CP/OL]. Alberta:Molecular Biology and Biotechnology Centre,University of Alberta,2000.
[8]	PEAKALL R, SMOUSE P E.GenAlEx 6.5:genetic analysis in Excel. Population genetic software for teaching and research-an update[J]. Bioinformatics,2012,28(19):2537-2539.
[9]	ROHLF F J. NTSYS-pc. Numerical Taxonomy and Multi-variate Analysis System,Version 2.1[CP/OL]. Setauket,New York:Exeter Software,2000.
[10]	NYBOM H. Comparison of different nuclear DNA markers for estimating intraspecific genetic diversity in plants[J].Mol Ecol,2004,13(5):1143-1155.
[11]	SONG Congwen,BAO Manzhu. Study on genetic diversity of RAPD mark for natural Davidia involucrata population[J]. Sci Silv Sin,2004,24(4):75-79.
[12]	LI Xueping,LI Zailiu,HE Chunling,et al. Genetic diversity of the endangered Davidia involucrata by AFLP analysis[J]. Acta Hortic Sin,2012,39(5):992-998.
[13]	ZHANG Rui,ZHOU Zhiguo,JIN Guoqing,et al. Genetic diversity and genetic differentiation of Taxus wallichiana var. mairei provenances[J]. Sci Silv Sin,2009,45(1):50-56.
[14]	ZHANG Junhong,HUANG Huahong,TONG Zaikang,et al. Genetic diversity in six natural populations of Betula luminifera from southern China[J]. Biodiversity Sci,2010,18(3):233-240.
[15]	HAMRICK J L,GODT M J W. Conservation genetics of endemic plant species[G]//AVISE J C,HAMRICK J L.Conservation Genetics:Case Histories from Nature. New York:Chapman and Hall,1996:281-304.
[16]	ISHIHAMA F,UENO S,TSUMURA Y,et al. Gene flow and inbreeding depression inferred from fine-scale genetic structure in an endangered heterostylous perennial,Primula sieboldi[J]. Mol Ecol,2005,14(4):983-990.
[17]	BARTISH I V,JEPPSSON N,NYBOM H. Population genetic structure in the dioecious pioneer plant species Hippophae rhamnoides investigated by random amplified polymorphic DNA(RAPD)markers[J]. Mol Ecol,1999,8(5):791-802.
[18]	NYBOM H,BARTISH I V. Effects of life history traits and sampling strategies on genetic diversity estimates obtained with RAPD markers in plants[J]. Evol Syst,2000,3(2):93-114.
[19]	YAN Maofen,LI Xianghua,WANG Kejing. Evaluation of genetic diversity by SSR markers for natural populations of wild soyban (Glycine soja) growing in the region of Beijing,China[J]. J Plant Ecol,2008,32(4):938-950.
[20]	WU Huifang,LI Zuozhou,HUANG Hongwen. Genetic differentiation among natural populations of Gastrodia elata (Orchidaceae) in Hubei and germplasm assessment of the cultivated populations[J]. Biodiversity Sci,2006,14(4):315-326.
[21]	CHEN Lianghua,HU Tingxing,ZHANG Fan,et al. Genetic diversity of four Juglans populations revealed by AFLP in Sichuan Province,China[J]. J Plant Ecol,2008,32(6):1362-1372.
[22]	LI Nan,LIU Xinhong,LI Yingang,et al. Genetic diversity in natural populations of Styrax tonkinensis[J]. Sci Silv Sin,2012,48(11):49-56.
[23]	CHEN Xiaoyong. Effects of habitat fragmentation on genetic structure of plant populations and implications for the biodiversity conservation[J]. Acta Ecol Sin,2000,20(5):885-892.

Proportional views

通讯作者: 陈斌, bchen63@163.com

1.
沈阳化工大学材料科学与工程学院沈阳 110142

Figures(2) / Tables(4)

Get Citation

PDF

XML

Article views(3813) PDF downloads(621) Cited by()

Proportional views

HTML

桂花Osmanthus fragrans属木犀科Oleaceae木犀属Osmanthus,是中国十大传统名花之一,具有非常高的经济价值和观赏价值。目前,桂花在中国南岭以北至秦岭以南的广大亚热带地区均有大量应用,其栽培面积和栽植数量是中国园林苗木中最大的种类之一。随着2004年中国取得了桂花国际登录权,进一步拓展了桂花生产和销售的国际和国内市场,这就要求我们必须进行桂花种质的改良和创新,培育花大、芳香、色艳、抗性强的桂花新品种。野生桂花具丰富的性状变异和遗传变异,是栽培桂花的重要育种资源,然而野生资源开发过度,破坏严重,适生生境散失,分布范围急剧缩小,种群规模和数量锐减；而现存的野生桂花种群仅在湖南^[1-3]、福建^[4]、浙江^{[3, 5]}、江西^[6]等地被零星发现。目前,野生桂花资源的研究主要集中在野生资源的调查^{[3, 6]}、种群动态^{[1, 4]}、繁育系统^{[2, 5]}等方面,而遗传多样性等方面的研究报道较少。江西全南和福建长汀的2个桂花自然种群保存较好,隔离分布,具有代表性。本研究利用扩增片段长度多态性(amplified fragment length polymorphism,AFLP)分子标记技术对2个不同类型(衰退型和稳定型)野生桂花种群遗传多样性进行分析,从而明确这2个种群的遗传结构,为桂花野生资源的科学保护和有效利用提供理论依据。

1. 材料与方法

1.1. 材料

研究对象为江西全南和福建长汀的2个桂花自然种群。前者位于江西省赣州市全南县,地理位置24.728°N,114.427°E,海拔约为342 m,为南岭北端,属中亚热带季风型气候,年平均气温为18.8 ℃；此处为一风水林,面积较小,仅有23株成龄桂花组成,地径大小为8～45 cm；林下无种子幼苗和天然压条形成的小苗,无自然更新能力,属于衰退型种群。后者位于福建省龙岩市长汀县,地理位置25.543°N,116.533°E,海拔约479 m,为武夷山脉南段,属中亚热带海洋性季风气候,年均气温18.7 ℃；此处为一孤岛,主要由石灰岩构成,面积较大,以灌木型和小乔木型桂花组成；灌木型桂花主要位于较低海拔区且土层较薄,小乔木型桂花处于地势较平坦处且土层相对较厚；林下存在较多天然更新幼苗,属于稳定型种群。江西全南共有23份样本,全部采集,所有材料地径均大于8 cm；福建长汀共采集73份样本,采集的材料地径均大于3 cm,相互间隔大于2 m。将采集的新鲜叶片放入硅胶干燥后带回实验室,置于-20 ℃冰箱中备用。

1.2. 方法

1.2.1. DNA提取和AFLP分析

本实验采用改进的十六烷基三甲基溴化铵(CTAB)法提取桂花基因组DNA,并用NanoDrop微量分光光度计(ND-1000)和8.0 g·L^-1琼脂糖凝胶电泳(六一电泳仪)检测总DNA的浓度与纯度,于-70 ℃保存备用。采用EcoRΙ和MseΙ双酶切组合以及从生工生物工程(上海)股份有限公司合成的64对引物中筛选出的7对引物组合进行AFLP分析。基本步骤如下：①酶切：300 ng DNA模板,50 nkat Eco RΙ,50 nkat MseΙ,1×NEB 缓冲液4,3 μg 牛血清白蛋白(BSA),补足双蒸水至30 μL。37 ℃恒温4 h,反应结束后75 ℃水浴15 min使酶失活。②连接：66.7 nkat T4 DNA ligast,1×T4 DNA 缓冲液,20 pmol E1-E2接头,20 pmol M1-M2接头,酶切产物10 μL,补足双蒸水至20 μL。16 ℃恒温10 h以上。③预扩增：1 μL连接产物,16.7 nkat Taq,1×缓冲液,4 nmol 三磷酸碱基脱氧核苷酸(dNTP),6 pmol EcoRⅠ预扩增引物,6 pmol MseⅠ预扩增引物,补足双蒸水至20 μL。反应条件为94 ℃,5 min；94 ℃,30 s,56 ℃,1 min,72 ℃,1 min,共25个循环；72 ℃ 10 min。预扩增产物稀释30倍,作为选择性扩增模板。④选择性扩增：预扩增释稀产物4 μL,16.7 nkat Taq,1×缓冲液,4 nmol dNTP,6 pmol EcoRⅠ选择性引物,6 pmol MseⅠ选择性引物,补足双蒸水至20 μL。反应条件为：94 ℃,5 min；94 ℃,30 s,65 ℃,30 s,72 ℃,1 min,共13循环(每循环降0.7 ℃)；94 ℃,30 s,56 ℃,30 s ,72 ℃,1 min,共25循环；72 ℃,7 min。扩增在Bio-RAD S1000TM Thermal Cycler PCR仪中进行。选择性扩增产物用60.0 g·L^-1变性聚丙烯酰胺凝胶电泳检测(Sequi-GenGT核酸测序电泳系统)。60 W恒功率预电泳30 min；6 μL选择性扩增产物与1 μL上样缓冲液[体积分数为98%甲酰胺,10 mmol·L^-1乙二胺四乙酸(EDTA),质量分数为0.25%二甲苯青FF,质量分数为0.25%溴酚蓝]混合,94 ℃变性5 min,结束后迅速置于冰上直到点样；加样7 μL·泳道^-1,70 W恒功率电泳,至二甲苯青FF至玻璃板2/3处,结束电泳；弱碱法银染显影,用Canon D60拍照保存图像。

1.2.2. 数据统计与分析

记录长度在500 bp以下清晰、稳定的扩增带,用“1-0”系统记录谱带,有条带的记为1,无条带的记为0,构成表型数据矩阵。AFLP分子标记为显性标记,因此假设分析的各位点都处于Hardy-Weinberg平衡。应用Popgen version 1.32软件^[7]分别为多态条带百分率(PPB),观测等位基因数(N_a)和有效等位基因数(N_e),Nei’s基因多样性指数(H_e),Shannon 多态性信息指数(I),物种的基因多样性(Ht),种群内的基因多样性(Hs)和种群间的遗传分化系数(G_st),基因流[N_m=0.5(1-G_st)/G_st]等遗传多样性参数。用GenAlex 6.5 软件^[8]进行分子方差分析(AMOVA)进行,计算物种的遗传变异以及遗传分化系数(PhiPT)以揭示群体遗传分量；用NTSYS-pc 2.10e软件^[9]对个体进行非加权配对算术平均法(UPGMA)聚类分析,估测群体遗传结构。

3. 讨论

物种的遗传多样性水平与其繁育系统、种群结构和地理分布格局等密切相关。从Nei’s遗传多样性指数和Shannon信息指数来看,桂花的相关遗传参数(H_e=0.285 6)均高于通过随机扩增多态性DNA标记(RAPD),AFLP和简单重复间序列(ISSR)等3种显性标记统计的多种植物的遗传多样性平均水平(H_e=0.22或0.23)^[10],且高于25种广布植物的平均水平(H_e=0.220 0)^[10],但低于珙桐Davidia involucrata(H_e=0.333 6或0.342 9)[11-12],南方红豆杉Taxus chinensis var. mairei(H_e=0.419 2)^[13]和光皮桦Betula luminifera(H_e=0.361 6)^[14],表明在种水平具有较高的遗传多样性。

植物种群的遗传结构不仅取决于自身的遗传基础、交配系统等,还受到遗传漂变、基因流和自然选择等因素影响^[15-17]。桂花遗传变异主要存在于种群内部,种群间的遗传分化系数(G_st)为0.161 6,低于基于AFLP标记的12个物种的平均水平(G_st=0.210 0)^[10],也低于9个广布物种的平均水平(G_st=0.330 0)^[10],而高于光皮桦(G_st=0.065 0)^[14]及南方红豆杉(G_st=0.121 1)^[13]。这表明桂花不同种群间遗传分化不显著,种群间存在一定的基因流,保证种群间遗传信息的交换,从而维持物种水平较高的遗传多样性。

福建长汀种群的遗传多样性程度明显高于江西全南种群,这与种群结构等有着密切的关系。福建长汀种群(稳定型)中存在世代更新现象,有利于种群内不同世代间遗传信息的传递以及遗传变异的产生、维持甚至增加；而缺少世代更新能力的江西全南种群(衰退型)只能维持原有的遗传多样性水平,甚至由于人为破坏造成种群结构和规模的变化,从而导致遗传多样性的下降。此外,不同的种群面积大小表现出不同的遗传差异^[18],往往面积较小种群的遗传多样性程度比面积较大的种群低,而与样本的数量没有明显的关系^[19-22]。本研究结果同样也支持这一观点。

Reference (23)

[1]	王贤荣, 袁发银, 陈昕. 湖南浏阳桂花峡桂花次生林的物种数量特征[J]. 南京林业大学学报:自然科学版, 2004, 28(supp): 41-44.	WANG Xianrong, YUAN Fayin, CHEN Xin. Quantitative characteristics of species in wild community of Osmanthus fragrans in Guihuaxia,Liuyang,Hunan[J]. J Nanjing For Univ Nat Sci Ed, 2004, 28(supp): 41-44.
[2]	汪德娥, 王宗海. 庐山山南桂花结实习性及野生群落研究[J]. 林业科技通讯, 1997, (10): 18-21.	WANG De'e, WANG Zonghai. Study on breeding and wild population of Osmanthus fragrans in south slopes of Lushan[J]. For Sci Technol, 1997, (10): 18-21.
[3]	雷瑞虎, 喻勋林, 王莉青. 中国野生桂花群落研究现状[J]. 安徽农业科学, 2008, 36(6): 2337-.	LEI Ruihu, YU Xunlin, WANG Liqing. Research on the introduction of Osmanthus fragrans community in China[J]. J Anhui Agric Sci, 2008, 36(6): 2337-.
[4]	董建文, 范小明, 吴东来. 福建长汀石峰寨景区桂花次生林群落物种数量特征[J]. 植物资源与环境学报, 2002, 11(4): 40-44.	DONG Jianwen, FAN Xiaoming, WU Donglai. Quantitative characteristics of species in secondary forest community of Osmanthus fragrans in Shifengzhai of Changting County,Fujian Province[J]. J Plant Resour Environ, 2002, 11(4): 40-44.
[5]	郝日明, 赵宏波, 王金虎. 野生桂花繁育系统的观察和研究[J]. 植物资源与环境学报, 2011, 20(1): 17-24.	HAO Riming, ZHAO Hongbo, WANG Jinhu. Observation and study on breeding system of wild Osmanthus fragrans[J]. J Plant Resour Environ, 2011, 20(1): 17-24.
[6]	段一凡, 谭志明, 刘远标. 江西全南古桂和野生桂花资源调查[J]. 植物资源与环境学报, 2012, 21(2): 118-120.	DUAN Yifan, TAN Zhiming, LIU Yuanbiao. Resources investigation of ancient tree and wild tree of Osmanthus fragrans in Quannan County of Jiangxi Province[J]. J Plant Resour Environ, 2012, 21(2): 118-120.
[7]	YEH F C,YANG R C,BOYLE T B J,et al. PopGene32,Microsoft Windows-Based Freeware for Population Genetic Analysis Version 1.32[CP/OL]. Alberta:Molecular Biology and Biotechnology Centre,University of Alberta,2000.
[8]	PEAKALL R, SMOUSE P E. GenAlEx 6.5:genetic analysis in Excel. Population genetic software for teaching and research-an update[J]. Bioinformatics, 2012, 28(19): 2537-2539. doi: 10.1093/bioinformatics/bts460
[9]	ROHLF F J. NTSYS-pc. Numerical Taxonomy and Multi-variate Analysis System,Version 2.1[CP/OL]. Setauket,New York:Exeter Software,2000.
[10]	NYBOM H. Comparison of different nuclear DNA markers for estimating intraspecific genetic diversity in plants[J]. Mol Ecol, 2004, 13(5): 1143-1155. doi: 10.1111/mec.2004.13.issue-5
[11]	宋丛文, 包满珠. 天然珙桐群体的RAPD标记遗传多样性研究[J]. 林业科学, 2004, 24(4): 75-79.	SONG Congwen, BAO Manzhu. Study on genetic diversity of RAPD mark for natural Davidia involucrata population[J]. Sci Silv Sin, 2004, 24(4): 75-79.
[12]	李雪萍, 李在留, 贺春玲. 珙桐遗传多样性的AFLP分析[J]. 园艺学报, 2012, 39(5): 992-998.	LI Xueping, LI Zailiu, HE Chunling. Genetic diversity of the endangered Davidia involucrata by AFLP analysis[J]. Acta Hortic Sin, 2012, 39(5): 992-998.
[13]	张蕊, 周志国, 金国庆. 南方红豆杉种源遗传多样性和遗传分化[J]. 林业科学, 2009, 45(1): 50-56.	ZHANG Rui, ZHOU Zhiguo, JIN Guoqing. Genetic diversity and genetic differentiation of Taxus wallichiana var. mairei provenances[J]. Sci Silv Sin, 2009, 45(1): 50-56.
[14]	张俊红, 黄华宏, 童再康. 光皮桦6个南方天然群体的遗传多样性[J]. 生物多样性, 2010, 18(3): 233-240. doi: 10.3724/SP.J.1003.2010.233	ZHANG Junhong, HUANG Huahong, TONG Zaikang. Genetic diversity in six natural populations of Betula luminifera from southern China[J]. Biodiversity Sci, 2010, 18(3): 233-240. doi: 10.3724/SP.J.1003.2010.233
[15]	HAMRICK J L,GODT M J W. Conservation genetics of endemic plant species[G]//AVISE J C,HAMRICK J L.Conservation Genetics:Case Histories from Nature. New York:Chapman and Hall,1996:281-304.
[16]	ISHIHAMA F, UENO S, TSUMURA Y. Gene flow and inbreeding depression inferred from fine-scale genetic structure in an endangered heterostylous perennial,Primula sieboldi[J]. Mol Ecol, 2005, 14(4): 983-990. doi: 10.1111/j.1365-294X.2005.02483.x
[17]	BARTISH I V, JEPPSSON N, NYBOM H. Population genetic structure in the dioecious pioneer plant species Hippophae rhamnoides investigated by random amplified polymorphic DNA(RAPD)markers[J]. Mol Ecol, 1999, 8(5): 791-802. doi: 10.1046/j.1365-294X.1999.00631.x
[18]	NYBOM H, BARTISH I V. Effects of life history traits and sampling strategies on genetic diversity estimates obtained with RAPD markers in plants[J]. Evol Syst, 2000, 3(2): 93-114.
[19]	严茂粉, 李向华, 王克晶. 北京地区野生大豆种群SSR标记的遗传多样性评价[J]. 植物生态学报, 2008, 32(4): 938-950.	YAN Maofen, LI Xianghua, WANG Kejing. Evaluation of genetic diversity by SSR markers for natural populations of wild soyban (Glycine soja) growing in the region of Beijing,China[J]. J Plant Ecol, 2008, 32(4): 938-950.
[20]	吴会芳, 李作洲, 黄宏文. 湖北野生天麻的遗传分化及栽培天麻种质评价[J]. 生物多样性, 2006, 14(4): 315-326. doi: 10.1360/biodiv.060053	WU Huifang, LI Zuozhou, HUANG Hongwen. Genetic differentiation among natural populations of Gastrodia elata (Orchidaceae) in Hubei and germplasm assessment of the cultivated populations[J]. Biodiversity Sci, 2006, 14(4): 315-326. doi: 10.1360/biodiv.060053
[21]	陈良华, 胡庭兴, 张帆. 用AFLP技术分析四川核桃资源的遗传多样性[J]. 植物生态学报, 2008, 32(6): 1362-1372.	CHEN Lianghua, HU Tingxing, ZHANG Fan. Genetic diversity of four Juglans populations revealed by AFLP in Sichuan Province,China[J]. J Plant Ecol, 2008, 32(6): 1362-1372.
[22]	李楠, 柳新红, 李因刚. 白花树天然群体的遗传多样性[J]. 林业科学, 2012, 48(11): 49-56.	LI Nan, LIU Xinhong, LI Yingang. Genetic diversity in natural populations of Styrax tonkinensis[J]. Sci Silv Sin, 2012, 48(11): 49-56.
[23]	陈小勇. 生境片断化对植物种群遗传结构的影响及植物遗传多样性保护[J]. 生态学报, 2000, 20(5): 885-892.	CHEN Xiaoyong. Effects of habitat fragmentation on genetic structure of plant populations and implications for the biodiversity conservation[J]. Acta Ecol Sin, 2000, 20(5): 885-892.

引物组合	总扩增条带数	多态性条带数	多态性条带百分率/%
E-AGC/M-CTG	47	47	100.00
E-AGC/M-CTA	50	43	86.00
E-AGG/M-CTG	41	29	70.73
E-AGG/M-CTA	48	43	89.58
E-ACC/M-CAT	29	23	79.31
E-AAC/M-CTT	59	51	86.44
E-AAG/M-CTA	56	40	71.43
合计	330	276
平均	47.14	39.42	83.64

引物组合	全南		长汀
引物组合	特异性条带数	特异性条带百分率/%	特异性条带数	特异性条带百分率/%
E-AGC/M-CTG	6	12.77	13	27.66
E-AGC/M-CTA	0	0	21	42
E-AGG/M-CTG	1	2.44	5	12.2
E-AGG/M-CTA	2	4.17	20	41.67
E-ACC/M-CAT	0	0	0	0
E-AAC/M-CTT	0	0	5	8.47
E-AAG/M-CTA	3	5.36	8	14.29
合计	12	3.64	72	21.82
平均	1.71		10.29

种群	多态位点百分率/%	Shannon多态性信息指数	Nei’ s基因多样性	观测等位基因数	有效等位基因数
江西全南	48.48	0.263 1 ± 0.246 4	0.177 8 ± 0.179 7	1.484 8 ± 0.370 5	1.3090 ± 0.354 8
福建长汀	79.09	0.414 8 ± 0.257 1	0.277 6 ± 0.183 6	1.7909±0.4073	1.4714±0.3498
平均	63.79	0.339 0 ± 0.252 8	0.227 7 ± 0.181 7	1.637 9 ± 0.388 9	1.3902 ± 0.352 3
种水平	83.64	0.428 3 ± 0.293 3	0.285 6 ± 0.204 6	1.8364±0.5005	1.4873 ± 0.377 2

变异来源	自由度d_j	总分差SSD	均方差MSE	方差成分	变异百分率/%	P值
种群间	1	617.540	617.540	16.486	29	<0.001
种群内	94	3 842.064	40.873	40.873	71	<0.001
合计	95	4 459.604		57.359	100

Genetic structure of different natural Osmanthus fragrans populations based on AFLP method

DOI: 10.11833/j.issn.2095-0756.2014.02.009