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大花序桉(Eucalyptus. cloeziana F. Muell.)为桉属昆士兰桉亚属(Idiogenes L.D.Pryor & L.A.S.Johnson ex Brooker)树种,自然分布仅限于澳大利亚昆士兰州4个不连续的地理分布区,其木材黄褐色、材质重硬、纹理通直、具黑金条纹,是很好的锯材树种[1-3]。我国引种大花序桉始于1972年,生长较迅速,材质优良,如17年生大花序桉平均基本密度达0.706 g·cm-3[4],目前已在广西和福建等地广泛用于实木用材林的营建。
遗传多样性是生态系统多样性和物种多样性的基础,研究一个物种的遗传多样性有助于理解其遗传变异水平及其在不同分布区的分化。分子标记是以DNA多态性为基础的遗传标记,广泛用作遗传多样性研究的手段,其中,简单重复序列(SSR)标记具有共显性、多态性高、重复性好等优点,已应用于细叶桉(E. tereticornis Smith)[5]、粗皮桉(E. pellita F. Muell.)[6-7]等桉属树种的遗传多样性研究。对于大花序桉,国外学者曾利用同工酶进行了种源的遗传多样性分析[8],近几年也利用木材力学性质[9-10]、生长和适应性[11]等表型性状进行遗传变异研究,但基于DNA水平的遗传多样性鲜见报道。本研究利用SSR标记对大花序桉4个主要分布地区进行了遗传多样性分析,旨在揭示大花序桉群体的遗传变异规律和遗传分化水平,以期为遗传资源的保存和育种潜力的评估提供理论基础。
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试验材料基于广西壮族自治区林业科学研究院引种的大花序桉种源/家系试验林,试验育苗的种子采自澳大利亚昆士兰州4个主要分布地区[12]的119株母树(半同胞家系)(表 1),每株母树(家系)选取1株子代采集嫩叶。
表 1 大花序桉样品起源的基本信息
Table 1. Original locality of E.cloeziana samples tested
起源地区
Original region纬度(S)
Latitude经度(E)
Longitude家系数量/个
Family number北部沿海
Northern coastal15°~20° 145°~147° 41 北部内陆
Northern inland17°~18° 145°~146° 17 南部内陆
Southern inland25°~26° 150°~152° 7 南部近沿海
Southern subcoastal25°~27° 152°~153° 54 -
大花序桉DNA提取采用天根生化科技(北京)有限公司的植物基因组DNA提取试剂盒,用1.5%琼脂糖凝胶电泳检测其浓度和质量。从已发表的桉树SSR标记中筛选出多态性较好的14个(表 2)用于本研究,包括Zhou等(标记序号1~8)[13]、周长品等(标记序号9和10)[14]、He等(标记序号11)[15]、Brondani等(标记序号12~14)[16]。SSR标记的检测方法参照文献[17],PCR产物利用ABI3130xl遗传分析仪(Applied Biosystems,美国)检测。
表 2 SSR标记及其引物序列
Table 2. SSR markers and their primer sequences
序号
No.SSR标记
SSR marker前向引物序列
Forward primer sequence(5′-3′)后向引物序列
Reverse primer sequence(5′-3′)1 EUCeSSR0294n TGCTGATGTTCCCGCCTAC GGAAGCAAATCATCCAGCAC 2 EUCeSSR0952 AAAAGCAGAGCGACAGCG TTCGGCCATAAACCATCC 3 EUCeSSR1145n2 GGCCGGGGACAGAAACTC TGCTACGGGAGCGAAACC 4 EUCeSSR0850 TAAACTCCATTCTCCTCCAA CGTGAACCCACATTGCTC 5 EUCeSSR0035n GCTCCCTCCTGCGAGATTT CGCCGATGATGATGTACTTGAA 6 EUCeSSR0620 CCGCACTCCAAGGACCACA CAGTAGCCGCTGCTCAAA 7 EUCeSSR0599 CCGAGAAACGCAGGATGT CGGCGGAGTCGTAGGAAGT 8 EUCeSSR0276n GATGGTTGCCAGATGACT TCTCATCTCATCATTCACTAAA 9 EUCeSSR0984 TCTTACGCACCGTCCTCT AGCCATTTCGCTCCTTCC 10 EUCeSSR1087n AGGCACAACAAGGTAAAGC GAGGGCCAATCTAGCATC 11 EUCeSSR712 CATTCCTCCGAGCATTTCC GCGACAACAACGGAGACAA 12 Embra8 CACAACTAAAAATCAAAACCC AAAGAGCAGATTATTACAGAAGC 13 Embra40 AAAGTATCTTCACGCTTCAT TCCCAATCATGATCTTCAG 14 Embra100 TGTGTTCTCGGTTTCAAAACT TGTGAAGTGATGCGAAGC -
利用软件GeneMarker V1.95[18]读取SSR标记数据,利用软件POPGEN1.31[19]计算各参数,利用软件NTSYSpc2.1[20]构建聚类树状图。
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14个SSR标记的多态性参数(表 3)表明:14个SSR标记共检测到249个等位片段,平均每个标记的等位片段为18个,其中,最多的标记(EUCeSSR0850)为24个,最少的标记(EUCeSSR0952)为6个;有效等位片段数为1.972 5~11.265 4,平均为6.959 6;Shannon′s信息指数为0.991 7~2.709 3,平均为2.159 7;Nei′s遗传多样性指数为0.493 0~0.911 2,平均为0.822 3;观测杂合度为0.273 5~0.769 2,平均为0.508 3;期望杂合度为0.495 1~0.915 1,平均为0.825 8。
表 3 14个SSR位点的遗传多样性参数
Table 3. Diversity parameters of the 14 SSR loci analyzed
SSR位点
SSR locus等位片段数(Na)
Number of
alleles有效等位片段数(Ne)
Effective number
of alleles观测杂合度(Ho)
Observed
heterozygosity期望杂合度(He)
Expected
heterozygosityShannon′s信息指数(I)
Shannon′s information
indexNei′s遗传多样性
指数(H)
Nei′s gene diversityEUCeSSR0294n 23 10.137 6 0.466 1 0.905 2 2.545 4 0.901 4 EUCeSSR0952 6 1.972 5 0.769 2 0.495 1 0.991 7 0.493 0 EUCeSSR1145n2 22 7.915 9 0.567 8 0.877 4 2.409 2 0.873 7 EUCeSSR0850 24 11.265 4 0.508 5 0.915 1 2.709 3 0.911 2 EUCeSSR0035n 11 4.660 1 0.487 2 0.788 8 1.758 9 0.785 4 EUCeSSR0620 18 5.274 1 0.273 5 0.813 9 2.021 6 0.810 4 EUCeSSR0599 17 5.302 4 0.440 7 0.814 9 2.064 7 0.811 4 EUCeSSR0276n 22 5.221 8 0.516 9 0.811 9 2.064 3 0.808 5 EUCeSSR0984 7 3.536 7 0.415 3 0.720 3 1.443 1 0.717 3 EUCeSSR1087n 14 8.027 7 0.627 1 0.879 2 2.243 2 0.875 4 EUCeSSR712 22 8.926 0 0.603 4 0.891 8 2.551 2 0.888 0 Embra8 23 6.982 9 0.550 8 0.860 4 2.396 6 0.856 8 Embra40 21 8.426 0 0.542 4 0.885 1 2.509 3 0.881 3 Embra100 19 9.785 0 0.347 5 0.901 6 2.527 9 0.897 8 均值
Mean18 6.959 6 0.508 3 0.825 8 2.159 7 0.822 3 -
大花序桉4个主要分布区的遗传多样性参数(表 4)表明:等位片段数为5.071 4~12.642 9,平均为9.982 1;有效等位片段数为3.751 6~6.526 7,平均为5.267 8;Shannon′s信息指数为1.404 2~1.966 4,平均为1.785 4;Nei′s遗传多样性指数为0.697 4~0.795 7,平均为0.761 8;观测杂合度为0.500 0~0.530 4,平均为0.510 0;期望杂合度为0.760 8~0.821 4,平均为0.788 2,表明各地区的多样性水平较高。大花序桉4个地区的遗传多样性参数存在差异,北部地区各参数值相对较大,可能是起源中心。
表 4 大花序桉的遗传多样性参数
Table 4. Genetic diversity parameters for the four original regions of E.cloeziana
起源地区
Original region等位片
段数(Na)
Number of
alleles有效等位片
段数(Ne)
Effective number
ofalleles观测杂
合度(Ho)
Observed
heterozygosity期望杂
合度(He)
Expected
heterozygosityShannon′s信息
指数(I)
Shannon′s
information indexNei′s遗传多样性
指数(H)
Nei′s gene
diversity北部沿海
Northern coastal12.642 9 6.044 2 0.502 6 0.793 2 1.966 4 0.783 7 北部内陆
Northern inland9.857 1 6.526 7 0.530 4 0.821 4 1.930 0 0.795 7 南部内陆
Southern inland5.071 4 3.751 6 0.500 0 0.760 8 1.404 2 0.697 4 南部近沿海
Southern subcoastal12.357 1 4.748 5 0.507 0 0.777 5 1.841 1 0.770 3 均值
Mean9.982 1 5.267 8 0.510 0 0.788 2 1.785 4 0.761 8 -
大花序桉4个主要分布区的遗传分化系数和基因流(表 5)表明:地区间遗传分化系数为0.025 8~0.106 7,平均为0.071 6;基因流为2.092 0~9.423 0,平均为3.242 9,说明大花序桉具有中等的遗传分化,且基因流动程度相对较高。
表 5 大花序桉遗传分化系数和基因流
Table 5. Coefficient of genetic differentiation and gene flow between E. cloeziana original regions
SSR位点
SSR locus地区内近交系数
Inbreeding coefficients of individuals
relative to the sub-region(Fis)地区间近交系数
Inbreeding coefficients of individuals
relative to the total region(Fit)地区间分化系数
Between-region
differentiation(Fst)基因流
Number of
migrants(Nm)EUCeSSR0294n 0.357 7 0.396 8 0.060 9 3.852 9 EUCeSSR0952 0.526 7 0.547 1 0.043 2 5.537 9 EUCeSSR1145n2 0.488 6 0.517 1 0.055 7 4.236 5 EUCeSSR0850 0.394 9 0.429 1 0.056 5 4.176 2 EUCeSSR0035n 0.289 8 0.324 1 0.048 3 4.928 5 EUCeSSR0620 0.137 9 0.196 7 0.068 2 3.413 1 EUCeSSR0599 0.162 7 0.252 0 0.106 7 2.092 0 EUCeSSR0276n 0.411 0 0.426 3 0.025 8 9.423 0 EUCeSSR0984 0.158 8 0.248 3 0.106 4 2.099 5 EUCeSSR1087n 0.602 4 0.619 1 0.041 8 5.726 9 EUCeSSR712 0.394 1 0.453 3 0.097 6 2.312 2 Embra8 0.358 8 0.409 6 0.079 2 2.907 4 Embra40 0.445 4 0.503 1 0.103 9 2.156 3 Embra100 0.228 3 0.303 8 0.097 8 2.305 0 均值
Mean0.355 4 0.401 6 0.071 6 3.242 9 分子方差(AMOVA) (表 6)表明:大部分遗传变异发生在地区内(93.2%),地区间方差分量仅占6.8%,表明大花序桉的群体结构较弱,群体间遗传分化不明显。
表 6 大花序桉遗传变异的AMOVA分析
Table 6. AMOVA results for within-and among-region variations in E. cloeziana
变异来源
Source of variation自由度
Degrees of freedom平方和
Sum of squares方差分量
Variance component方差分量比
Ratio of variance/%地区间
Among regions3 92.4 0.440 4 6.8 地区内个体间
Among individuals within regions115 944.5 2.266 3 35.5 个体间
Within individuals119 438.0 3.680 7 57.7 总计
Total237 1 474.9 6.387 3 100.0 4个地区间的遗传一致度和遗传距离(表 7)表明:遗传一致度为0.649 8~0.896 1,平均为0.717 2;遗传距离为0.109 8~0.431 0,平均为0.338 9。基于遗传一致度,利用非加权分组算术平均(UPGMA)法进行聚类分析(图 1)表明:遗传一致度为0.74时,大花序桉4个主要分布区被划分为北部亚类和南部亚类。
表 7 大花序桉4个地区间的Nei′s遗传一致度与遗传距离
Table 7. Nei′s genetic identity and genetic distance between E. cloeziana original regions
起源地区
Original region北部沿海
Northern coastal北部内陆
Northern inland南部内陆
Southern inland南部近沿海
Southern subcoastal北部沿海
Northern coastal0.896 1 0.675 9 0.649 8 北部内陆
Northern inland0.109 8 0.669 0 0.665 9 南部内陆
Southern inland0.391 7 0.402 0 0.746 4 南部近沿海
Southern subcoastal0.431 0 0.406 6 0.292 5 注:对角线上方为遗传一致度,对角线下方为遗传距离。
Note: The genetic identity was above the diagonal, and the genetic distance was below the diagonal.
大花序桉的遗传多样性分析
Genetic Diversity of Eucalyptus cloeziana
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摘要:
目的 揭示大花序桉的遗传多样性,为大花序桉群体资源的保存和育种潜力的评估提供理论基础。 方法 利用14个简单重复序列(SSR)标记对大花序桉4个主要分布地区进行变异检测,分析位点多态性和群体多样性,计算地区间的分化系数和遗传相似性以及地区间和地区内的分子方差分量,基于遗传相似性进行聚类分析。 结果 14个SSR标记共检测到249个等位片段,平均每个标记检测到18个等位片段。基于所有标记,大花序桉各地区的Shannon's信息指数平均为1.785 4,观测杂合度平均为0.510 0,期望杂合度平均为0.788 2,表明遗传多样性较高。地区间平均遗传分化系数为0.071 6,分子方差分析(AMOVA)中群体间方差分量仅为6.8%,表明遗传分化水平中等、遗传变异主要存在于群体内。非加权分组算术平均法(UPGMA)聚类分析将大花序桉4个主要分布地区划分为北部和南部2大类。 结论 种质资源保存要优先考虑多样性较高的地区。大花序桉遗传多样性较高,进一步选育的潜力较大。 Abstract:Objective To study the genetic diversity of Eucalyptus cloeziana for the purposes of germplasm conservation and breeding potential evaluation. Method Fourteen simple sequence repeats (SSR) markers were used to detect the variation in four main distribution areas of E. cloeziana. The locus polymorphism and population diversity were analyzed, the differentiation coefficient and genetic similarity among regions and the molecular variance components inter-and intra-regions were calculated. Cluster analysis was conducted based on genetic similarity. Result A total of 249 alleles were detected in 14 SSR markers, with an average of 18 alleles per marker. Based on all the markers, the Shannon's information index was 1.785 4, the observed heterozygosity was 0.510 0, and the expected heterozygosity was 0.788 2, indicating a high genetic diversity. The average coefficient of genetic differentiation among regions was 0.071 6, and the component of variance among populations in analysis of molecular variance (AMOVA) was only 6.8%. This indicated that the level of genetic differentiation was moderate and genetic variation mainly exists in populations. Unweighted pair group method with arithmetic (UPGMA) clustering analysis divided the four main distribution areas of E. cloeziana into two major categories:north and south. Conclusion The germplasm resource conservation should give priority to the areas with high diversity. The genetic diversity of E. cloeziana is high, and it has great potential for breeding. -
Key words:
- Eucalyptus cloeziana
- / SSR
- / genetic diversity
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表 1 大花序桉样品起源的基本信息
Table 1. Original locality of E.cloeziana samples tested
起源地区
Original region纬度(S)
Latitude经度(E)
Longitude家系数量/个
Family number北部沿海
Northern coastal15°~20° 145°~147° 41 北部内陆
Northern inland17°~18° 145°~146° 17 南部内陆
Southern inland25°~26° 150°~152° 7 南部近沿海
Southern subcoastal25°~27° 152°~153° 54 表 2 SSR标记及其引物序列
Table 2. SSR markers and their primer sequences
序号
No.SSR标记
SSR marker前向引物序列
Forward primer sequence(5′-3′)后向引物序列
Reverse primer sequence(5′-3′)1 EUCeSSR0294n TGCTGATGTTCCCGCCTAC GGAAGCAAATCATCCAGCAC 2 EUCeSSR0952 AAAAGCAGAGCGACAGCG TTCGGCCATAAACCATCC 3 EUCeSSR1145n2 GGCCGGGGACAGAAACTC TGCTACGGGAGCGAAACC 4 EUCeSSR0850 TAAACTCCATTCTCCTCCAA CGTGAACCCACATTGCTC 5 EUCeSSR0035n GCTCCCTCCTGCGAGATTT CGCCGATGATGATGTACTTGAA 6 EUCeSSR0620 CCGCACTCCAAGGACCACA CAGTAGCCGCTGCTCAAA 7 EUCeSSR0599 CCGAGAAACGCAGGATGT CGGCGGAGTCGTAGGAAGT 8 EUCeSSR0276n GATGGTTGCCAGATGACT TCTCATCTCATCATTCACTAAA 9 EUCeSSR0984 TCTTACGCACCGTCCTCT AGCCATTTCGCTCCTTCC 10 EUCeSSR1087n AGGCACAACAAGGTAAAGC GAGGGCCAATCTAGCATC 11 EUCeSSR712 CATTCCTCCGAGCATTTCC GCGACAACAACGGAGACAA 12 Embra8 CACAACTAAAAATCAAAACCC AAAGAGCAGATTATTACAGAAGC 13 Embra40 AAAGTATCTTCACGCTTCAT TCCCAATCATGATCTTCAG 14 Embra100 TGTGTTCTCGGTTTCAAAACT TGTGAAGTGATGCGAAGC 表 3 14个SSR位点的遗传多样性参数
Table 3. Diversity parameters of the 14 SSR loci analyzed
SSR位点
SSR locus等位片段数(Na)
Number of
alleles有效等位片段数(Ne)
Effective number
of alleles观测杂合度(Ho)
Observed
heterozygosity期望杂合度(He)
Expected
heterozygosityShannon′s信息指数(I)
Shannon′s information
indexNei′s遗传多样性
指数(H)
Nei′s gene diversityEUCeSSR0294n 23 10.137 6 0.466 1 0.905 2 2.545 4 0.901 4 EUCeSSR0952 6 1.972 5 0.769 2 0.495 1 0.991 7 0.493 0 EUCeSSR1145n2 22 7.915 9 0.567 8 0.877 4 2.409 2 0.873 7 EUCeSSR0850 24 11.265 4 0.508 5 0.915 1 2.709 3 0.911 2 EUCeSSR0035n 11 4.660 1 0.487 2 0.788 8 1.758 9 0.785 4 EUCeSSR0620 18 5.274 1 0.273 5 0.813 9 2.021 6 0.810 4 EUCeSSR0599 17 5.302 4 0.440 7 0.814 9 2.064 7 0.811 4 EUCeSSR0276n 22 5.221 8 0.516 9 0.811 9 2.064 3 0.808 5 EUCeSSR0984 7 3.536 7 0.415 3 0.720 3 1.443 1 0.717 3 EUCeSSR1087n 14 8.027 7 0.627 1 0.879 2 2.243 2 0.875 4 EUCeSSR712 22 8.926 0 0.603 4 0.891 8 2.551 2 0.888 0 Embra8 23 6.982 9 0.550 8 0.860 4 2.396 6 0.856 8 Embra40 21 8.426 0 0.542 4 0.885 1 2.509 3 0.881 3 Embra100 19 9.785 0 0.347 5 0.901 6 2.527 9 0.897 8 均值
Mean18 6.959 6 0.508 3 0.825 8 2.159 7 0.822 3 表 4 大花序桉的遗传多样性参数
Table 4. Genetic diversity parameters for the four original regions of E.cloeziana
起源地区
Original region等位片
段数(Na)
Number of
alleles有效等位片
段数(Ne)
Effective number
ofalleles观测杂
合度(Ho)
Observed
heterozygosity期望杂
合度(He)
Expected
heterozygosityShannon′s信息
指数(I)
Shannon′s
information indexNei′s遗传多样性
指数(H)
Nei′s gene
diversity北部沿海
Northern coastal12.642 9 6.044 2 0.502 6 0.793 2 1.966 4 0.783 7 北部内陆
Northern inland9.857 1 6.526 7 0.530 4 0.821 4 1.930 0 0.795 7 南部内陆
Southern inland5.071 4 3.751 6 0.500 0 0.760 8 1.404 2 0.697 4 南部近沿海
Southern subcoastal12.357 1 4.748 5 0.507 0 0.777 5 1.841 1 0.770 3 均值
Mean9.982 1 5.267 8 0.510 0 0.788 2 1.785 4 0.761 8 表 5 大花序桉遗传分化系数和基因流
Table 5. Coefficient of genetic differentiation and gene flow between E. cloeziana original regions
SSR位点
SSR locus地区内近交系数
Inbreeding coefficients of individuals
relative to the sub-region(Fis)地区间近交系数
Inbreeding coefficients of individuals
relative to the total region(Fit)地区间分化系数
Between-region
differentiation(Fst)基因流
Number of
migrants(Nm)EUCeSSR0294n 0.357 7 0.396 8 0.060 9 3.852 9 EUCeSSR0952 0.526 7 0.547 1 0.043 2 5.537 9 EUCeSSR1145n2 0.488 6 0.517 1 0.055 7 4.236 5 EUCeSSR0850 0.394 9 0.429 1 0.056 5 4.176 2 EUCeSSR0035n 0.289 8 0.324 1 0.048 3 4.928 5 EUCeSSR0620 0.137 9 0.196 7 0.068 2 3.413 1 EUCeSSR0599 0.162 7 0.252 0 0.106 7 2.092 0 EUCeSSR0276n 0.411 0 0.426 3 0.025 8 9.423 0 EUCeSSR0984 0.158 8 0.248 3 0.106 4 2.099 5 EUCeSSR1087n 0.602 4 0.619 1 0.041 8 5.726 9 EUCeSSR712 0.394 1 0.453 3 0.097 6 2.312 2 Embra8 0.358 8 0.409 6 0.079 2 2.907 4 Embra40 0.445 4 0.503 1 0.103 9 2.156 3 Embra100 0.228 3 0.303 8 0.097 8 2.305 0 均值
Mean0.355 4 0.401 6 0.071 6 3.242 9 表 6 大花序桉遗传变异的AMOVA分析
Table 6. AMOVA results for within-and among-region variations in E. cloeziana
变异来源
Source of variation自由度
Degrees of freedom平方和
Sum of squares方差分量
Variance component方差分量比
Ratio of variance/%地区间
Among regions3 92.4 0.440 4 6.8 地区内个体间
Among individuals within regions115 944.5 2.266 3 35.5 个体间
Within individuals119 438.0 3.680 7 57.7 总计
Total237 1 474.9 6.387 3 100.0 表 7 大花序桉4个地区间的Nei′s遗传一致度与遗传距离
Table 7. Nei′s genetic identity and genetic distance between E. cloeziana original regions
起源地区
Original region北部沿海
Northern coastal北部内陆
Northern inland南部内陆
Southern inland南部近沿海
Southern subcoastal北部沿海
Northern coastal0.896 1 0.675 9 0.649 8 北部内陆
Northern inland0.109 8 0.669 0 0.665 9 南部内陆
Southern inland0.391 7 0.402 0 0.746 4 南部近沿海
Southern subcoastal0.431 0 0.406 6 0.292 5 注:对角线上方为遗传一致度,对角线下方为遗传距离。
Note: The genetic identity was above the diagonal, and the genetic distance was below the diagonal. -
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