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映山红(Rhododendron simsii Planch.),又名杜鹃花、山石榴,属于杜鹃花科(Ericaceae)杜鹃花属(Rhododendron)植物,具有极高的观赏价值和药用价值,是北温带森林生态系统中的主要成员[1-2]。研究映山红不同龄级种群的遗传多样性和遗传结构差异,探究影响因素,对映山红资源的可持续开发意义重大。
Wu等[3]采用AFLP显性标记对濒危物种大树杜鹃(R. protistum var. giganteum)的遗传多样性和遗传结构进行了研究。Liu等[4]采用ISSR和RAPD标记对长白山杜鹃(R. aureum Georgi)种群的遗传多样性和遗传结构进行了研究。基于随机引物,Kuttapetty等[5]发现树形杜鹃(R. arboreum)种群具有高的遗传多样性(Ht=0.21,Nm=1.13)。Zhao等[6]采用AFLP标记发现秀雅杜鹃(R. concinnum)种群在物种水平和种群水平上均有较高的遗传多样性。目前,对映山红的保护遗传学研究比较少,而且主要集中于空间尺度上,尚缺乏时间尺度上的研究。
微卫星标记(SSR)在基因组中广泛分布、多态性丰富、重复性好、特异性强、共显性遗传,在植物遗传多样性研究中被广泛运用,但是在映山红的遗传多样性研究中应用较少[7]。本研究在映山红空间遗传多样性研究的基础上,基于前期RNA-seq技术开发的SSR标记阐述世代结构类型丰富的大别山黄狮寨映山红种群时间尺度上的遗传多样性,揭示不同世代遗传多样性的变化规律,从分子层面探讨映山红的生存潜力和进化方向,为有效保护大别山野生映山红资源提供理论依据。
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12个微卫星位点共检测到61个等位基因,每个位点为3!9个,等位基因大小为149~262 bp(表 1)。WRsE-37标记检测到9个等位基因(图 1),等位基因数最少的是WRsE-90和WRsE-97,仅检测到3个。在4个年龄级种群内分别检测到43、50、48、47个等位基因(表 2)。Shannon信息指数变化范围为1.025 0~1.188 5。Shannon信息指数反映的不同龄级映山红遗传多样性的顺序为:小树>成树>老树>幼苗。观察杂合度和期望杂合度的变化范围分别为0.676 4~0.881 2和0.607 7~0.690 7。Nei's基因多样度变化范围为0.578 2~0.654 4,平均为0.632 6,在小树种群内最大,最小的是幼苗种群,与Shannon信息指数反映的一致。4个种群均存在HO大于HE的情况,即杂合子过剩现象。经Bonferroni校正(P < 0.004),8个微卫星位点严重偏离Hardy-Weinberg平衡(表 3)。
表 1 微卫星标记信息
Table 1. Information of microsatellite markers
位点 Locus 正向引物(5′-3′)
Forward primer (5′-3′)反向引物(5′-3′)
Reverse primer (5′-3′)基序
Motif退火温度 Annealing temperature /℃ 片段大小
Size range /bpWRsE-37 CTTGCCACTTTGAGTTTGAG AGAGTAATTTGGAGGAAGCG (CT)28 55 200~212 WRsE-43 TGAACCCTTCTTCTTCTTCC TTTGATTGAAGGGTGGAGTG (TC)23 56 249~262 WRsE-70 TCTTCCGATTCCATCATTCC TGGGCGTGATTTGGTTATAA (CT)22 58 178~192 WRsE-77 TAGCTGCTTACTGTTGAAGG AGTAAAAGGGCTGAAACTGT (AG)22 57 149~165 WRsE-81 GCCCTATCCCTCAACTTTAC GAGGAGCGTGGTTAGTAATT (TC)21 56 229~253 WRsE-82 GTATGGGACCTGTGATTTCC CTCCAACTAGCTACTCCAAC (GA)24 57 228~236 WRsE-85 GAAATCTCGAATCACCTCCA AAGGTGTTGGTGGACTAATC (TC)21 56 149~167 WRsE-90 TTGAAGAACACTCAAGTTGC ACGTAGAACATTGCTTTCCT (GA)21 57 196~202 WRsE-93 GGTATCCGGTTTTCATCACT ATACCCACTAGCAACAGAGA (GA)23 57 233~256 WRsE-97 AGAAAACTGGGAGATGTGTC AGGTGATCATCTTTGCATGT (CT)21 58 222~246 WRsE-113 TATTGTACAGCTCCCCTTTG CCTCAATGTTCTATCGACGT (CT)23 57 185~207 WRsE-131 CTCTTTCCCCTTTCATGTGA AAGTGTTGAGTCCTCGTATG (TC)23 58 153~171 表 2 映山红种群4个不同年龄级的遗传多样性
Table 2. Genetic diversity of four different life stages of R. simsii
年龄级 Age group 样本数 Population size Na NE I HO HE h 幼苗 15 43 30.38 1.025 0 0.676 4 0.607 7 0.578 2 小树 15 50 36.52 1.188 5 0.881 2 0.690 7 0.654 4 成树 15 48 34.58 1.134 1 0.825 0 0.657 8 0.629 4 老树 15 47 35.77 1.133 7 0.846 3 0.672 8 0.632 6 注:Na:等位基因数;NE:有效等位基因数;I:Shannon多样性指数;HO:观察杂合度;HE:期望杂合度;h:Nei基因多样性指数。
Note: Na, NE, I, HO, HE, and h referred to number of alleles, effective number of alleles, Shannon's information index, observed heterozygosity, expected heterozygosity, and Nei's genetic diversity, respectively.表 3 映山红12个微卫星位点的遗传分化和基因流信息
Table 3. Summary of genetic variation and gene flow at 9 loci
位点 Locus Fis Fit Fst Nm HWE WRsE-37 -0.241 4 -0.156 9 0.068 1 3.419 3 0.000 0* WRsE-43 0.174 4 0.270 6 0.116 5 1.896 3 0.037 9 WRsE-70 -0.255 9 -0.169 7 0.068 6 3.393 4 0.001 9* WRsE-77 -0.073 8 -0.014 1 0.055 6 4.246 5 0.034 6 WRsE-81 -0.391 8 0.032 0 0.304 5 0.570 9 0.000 0* WRsE-82 -0.220 2 -0.104 5 0.094 8 2.387 3 0.000 2* WRsE-85 -0.408 3 -0.287 7 0.085 6 2.670 6 0.000 2* WRsE-90 -0.568 3 -0.534 3 0.021 7 11.271 0 0.001 1* WRsE-93 0.003 9 0.069 5 0.065 8 3.550 2 0.699 9 WRsE-97 -0.638 3 -0.615 1 0.014 2 17.404 7 0.000 0* WRsE-113 -0.561 3 -0.301 1 0.166 7 1.249 7 0.000 0* WRsE-131 -0.444 6 -0.247 4 0.136 5 1.581 6 0.007 1 平均 Mean -0.294 6 -0.162 1 0.102 4 2.192 4 注:*标注的是严重偏离Hardy-Weinberg平衡的位点。Fis:种群内近交系数;Fit:总近交系数;Fst:遗传分化系数;Nm:基因流、HWE:Hardy-Weinberg平衡
Note: * referring to locus significantly deviating from Hardy-Weinberg equilibrium. Fis, Fit, Fst, Nm, and HWE referred to intra-population inbreeding coefficient, total-population inbreeding coefficient, inter-population genetic differentiation coefficient (Fst), gene flow, and Hardy-Weinberg equilibrium, respectively -
种群内近交系数Fis变化范围为-0.638 3~0.174 4,平均值为-0.290 6;总近交系数Fit变化范围为-0.615 1~0.270 6,平均值为-0.162 1(表 3)。依据Nagylaki等[12]的理论,Fis和Fit平均值均为负数,表明映山红种群在检测的12个微卫星位点杂交现象严重,各种群以杂合子为主,尤其是WRsE-90、WRsE-97、WRsE-113位点。然而,在WRsE-43(Fis=0.174 4; Fit=0.270 6)和WRsE-93(Fis=0.003 9; Fit=0.069 5)位点存在着近交现象。
遗传分化系数Fst变化范围为0.014 2~0.304 5,平均值为0.102 4,表明仅10.24%的遗传变异发生在种群间,发生在种群内的遗传变异高达89.76%(表 3)。吕召云等[13]认为,Fst大于0.25时种群极度分化,Fst值在0.15和0.25之间种群间明显分化,Fst值在0.05和0.15之间种群间中度分化,Fst小于0.05种群间几乎无分化。在检测的12个位点处,WRsE-81位点极度分化,WRsE-113位点为明显分化,而WRsE-90和WRsE-97两个位点几乎无分化,其余8个位点为中度分化。
作为影响种群分化的重要因素,基因流Nm变化范围为0.570 9~17.404 7,平均值为2.192 4。WRsE-81(Nm=0.570 9)基因流水平非常低,种群在此位点很容易因遗传漂变而分化。其余11个微卫星位点的Nm值均大于1,足以抵抗种群遗传漂变导致的分化,尤其是杂交现象明显的位点WRsE-90(Nm=11.271 0)和WRsE-97 (Nm=17.404 7)。
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采用Nei氏方法计算4个世代的遗传距离(D)和遗传一致度(S),种群遗传一致度变化范围为0.583 1~0.867 6,遗传距离变化范围为0.142 0~0.563 9(表 4)。成树种群与小树种群的遗传一致度最大,其次是成树种群与老树种群之间,老树种群与幼苗种群的遗传一致度最小。相反,老树种群与幼苗种群之间的遗传距离最大,其次是成树与幼苗种群,遗传距离最小的是小树与成树种群。根据Nei's遗传距离,采用UPGMA方法构建聚类图,小树和成树种群先被聚在一起,然后与老树种群聚类在一个大分支,幼苗则独立被聚成一支(图 1)。
表 4 映山红种群不同年龄级间的Nei遗传一致度(右上)和遗传距离(左下)
Table 4. Genetic identify (above) and genetic distance (below) among different life stages of R. simsii population
幼苗
Seedlings小树
Juvenile成树
Adult老树
Old trees幼苗 Seedlings - 0.740 0 0.693 0 0.583 1 小树 Juvenile 0.301 2 - 0.867 6 0.766 7 成树 Adult 0.366 7 0.142 0 - 0.859 4 老树 Old trees 0.563 9 0.265 6 0.151 5 -
大别山不同龄级映山红种群遗传多样性的SSR分析
Genetic Diversity of Rhododendron simsii Populations on Dabieshan at Different Life Stages Based on SSR Markers
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摘要:
目的 利用SSR标记比较大别山黄狮寨不同年龄级映山红(Rhododendron simsii Planch.)种群的遗传多样性及遗传结构,探究映山红不同世代间遗传多样性的变化规律,为大别山野生映山红资源的合理利用和高效保护提供科学依据。 方法 按照基径大小和丛枝多少将大别山黄狮寨典型映山红种群划分为老树、成树、小树、幼苗4个年龄级,筛选出12对多态性强的SSR引物用于PCR扩增,扩增产物经6%变性聚丙烯酰胺凝胶电泳检测并银染。构建"0/1"矩阵,利用POPGENE 32.0软件分析种群遗传多样性。基于Nei's遗传距离,采用软件MEGA5.0进行UPGMA聚类。 结果 不同龄级的映山红遗传多样性差别较大,幼苗和老树种群多样性最差,小树种群多样性最丰富。12个微卫星标记观测等位基因数为3~9个,平均5.08;有效等位基因数为2.254 9~6.129 7,平均3.460 5;观察杂合度HO和期望杂合度HE分别为0.676 4~0.881 2和0.607 7~0.690 7。Shannon信息指数(I)以小树群体最高,成树次之,幼苗最低。近交系数Fis为-0.638 3~0.174 4,平均为-0.294 6;总近交系数Fit为-0.615 1~0.270 6,平均为-0.162 1,表明各龄级种群内主要繁殖方式为杂交。分子方差分析(AMOVA)表明89.76%的遗传变异存在于年龄级内,仅10.24%存在于年龄级间。基因流水平高,仅1个位点Nm < 1。遗传一致度最高的为小树和成树种群。 结论 大别山黄狮寨映山红种群遗传多样性丰富,种群间中度分化,遗传变异主要存在于年龄级内。 Abstract:Objective Microsatellite markers were used to compare the genetic diversity and genetic structure of Rhododendron simsii populations at four different life stages sampled from Huangshizhai of Dabieshan region to clarify the mechanism of genetic diversity among different age groups. Method The populations of R. simsii were divided into four age groups (old trees, adult, juvenile, and seedlings) according to their sizes of basal diameter and numbers of branches. Twelve SSR markers were used for PCR amplification, and the products were subjected to 6% polyacrylamide gel electrophoresis, which were further visualized through silver staining. The "0/1" matrix was constructed and POPGENE 32.0 software was used to analyze the genetic diversity. Based on Nei's genetic distance, UPGMA dendrogram was constructed with MEGA 5.0 software. Result Different genetic diversities were observed in the four R. simsii populations, the lowest genetic diversity was observed in seedlings population, followed by old trees population, while the highest genetic diversity was observed in juvenile population. The observed and effective number of alleles per locus ranged from 3 to 9 and 2.254 9-6.129 7, with the average of 5.08 and 3.460 5, respectively. Moreover, HO and HE were in the ranges of 0.676 4-0.881 2 and 0.607 7-0.690 7, respectively. The Shannon's information index (I) was the highest in juvenile population, followed by adult population, and lowest in seedling population. Fis and Fit ranged within-0.638 3-0.174 4 and-0.615 1-0.270 6, with the mean of-0.294 6 and-0.162 1, indicating the outcross took place frequently. In particular, the AMOVA analysis showed that 89.76% of genetic variation was maintained within age groups, while only 10.24% presented among age groups. Frequent gene flow was observed, as only one locus showed Nm less than 1.0.The genetic identify between adult and juvenile groups was the highest. Conclusion High genetic diversity existed among R. simsii populations located on Huangshizhai. Moderate differentiation was observed, which was maintained between age groups. -
Key words:
- Rhododendron simsii Planch.
- / life stage
- / genetic diversity
- / population structure
- / microsatellite
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表 1 微卫星标记信息
Table 1. Information of microsatellite markers
位点 Locus 正向引物(5′-3′)
Forward primer (5′-3′)反向引物(5′-3′)
Reverse primer (5′-3′)基序
Motif退火温度 Annealing temperature /℃ 片段大小
Size range /bpWRsE-37 CTTGCCACTTTGAGTTTGAG AGAGTAATTTGGAGGAAGCG (CT)28 55 200~212 WRsE-43 TGAACCCTTCTTCTTCTTCC TTTGATTGAAGGGTGGAGTG (TC)23 56 249~262 WRsE-70 TCTTCCGATTCCATCATTCC TGGGCGTGATTTGGTTATAA (CT)22 58 178~192 WRsE-77 TAGCTGCTTACTGTTGAAGG AGTAAAAGGGCTGAAACTGT (AG)22 57 149~165 WRsE-81 GCCCTATCCCTCAACTTTAC GAGGAGCGTGGTTAGTAATT (TC)21 56 229~253 WRsE-82 GTATGGGACCTGTGATTTCC CTCCAACTAGCTACTCCAAC (GA)24 57 228~236 WRsE-85 GAAATCTCGAATCACCTCCA AAGGTGTTGGTGGACTAATC (TC)21 56 149~167 WRsE-90 TTGAAGAACACTCAAGTTGC ACGTAGAACATTGCTTTCCT (GA)21 57 196~202 WRsE-93 GGTATCCGGTTTTCATCACT ATACCCACTAGCAACAGAGA (GA)23 57 233~256 WRsE-97 AGAAAACTGGGAGATGTGTC AGGTGATCATCTTTGCATGT (CT)21 58 222~246 WRsE-113 TATTGTACAGCTCCCCTTTG CCTCAATGTTCTATCGACGT (CT)23 57 185~207 WRsE-131 CTCTTTCCCCTTTCATGTGA AAGTGTTGAGTCCTCGTATG (TC)23 58 153~171 表 2 映山红种群4个不同年龄级的遗传多样性
Table 2. Genetic diversity of four different life stages of R. simsii
年龄级 Age group 样本数 Population size Na NE I HO HE h 幼苗 15 43 30.38 1.025 0 0.676 4 0.607 7 0.578 2 小树 15 50 36.52 1.188 5 0.881 2 0.690 7 0.654 4 成树 15 48 34.58 1.134 1 0.825 0 0.657 8 0.629 4 老树 15 47 35.77 1.133 7 0.846 3 0.672 8 0.632 6 注:Na:等位基因数;NE:有效等位基因数;I:Shannon多样性指数;HO:观察杂合度;HE:期望杂合度;h:Nei基因多样性指数。
Note: Na, NE, I, HO, HE, and h referred to number of alleles, effective number of alleles, Shannon's information index, observed heterozygosity, expected heterozygosity, and Nei's genetic diversity, respectively.表 3 映山红12个微卫星位点的遗传分化和基因流信息
Table 3. Summary of genetic variation and gene flow at 9 loci
位点 Locus Fis Fit Fst Nm HWE WRsE-37 -0.241 4 -0.156 9 0.068 1 3.419 3 0.000 0* WRsE-43 0.174 4 0.270 6 0.116 5 1.896 3 0.037 9 WRsE-70 -0.255 9 -0.169 7 0.068 6 3.393 4 0.001 9* WRsE-77 -0.073 8 -0.014 1 0.055 6 4.246 5 0.034 6 WRsE-81 -0.391 8 0.032 0 0.304 5 0.570 9 0.000 0* WRsE-82 -0.220 2 -0.104 5 0.094 8 2.387 3 0.000 2* WRsE-85 -0.408 3 -0.287 7 0.085 6 2.670 6 0.000 2* WRsE-90 -0.568 3 -0.534 3 0.021 7 11.271 0 0.001 1* WRsE-93 0.003 9 0.069 5 0.065 8 3.550 2 0.699 9 WRsE-97 -0.638 3 -0.615 1 0.014 2 17.404 7 0.000 0* WRsE-113 -0.561 3 -0.301 1 0.166 7 1.249 7 0.000 0* WRsE-131 -0.444 6 -0.247 4 0.136 5 1.581 6 0.007 1 平均 Mean -0.294 6 -0.162 1 0.102 4 2.192 4 注:*标注的是严重偏离Hardy-Weinberg平衡的位点。Fis:种群内近交系数;Fit:总近交系数;Fst:遗传分化系数;Nm:基因流、HWE:Hardy-Weinberg平衡
Note: * referring to locus significantly deviating from Hardy-Weinberg equilibrium. Fis, Fit, Fst, Nm, and HWE referred to intra-population inbreeding coefficient, total-population inbreeding coefficient, inter-population genetic differentiation coefficient (Fst), gene flow, and Hardy-Weinberg equilibrium, respectively表 4 映山红种群不同年龄级间的Nei遗传一致度(右上)和遗传距离(左下)
Table 4. Genetic identify (above) and genetic distance (below) among different life stages of R. simsii population
幼苗
Seedlings小树
Juvenile成树
Adult老树
Old trees幼苗 Seedlings - 0.740 0 0.693 0 0.583 1 小树 Juvenile 0.301 2 - 0.867 6 0.766 7 成树 Adult 0.366 7 0.142 0 - 0.859 4 老树 Old trees 0.563 9 0.265 6 0.151 5 - -
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