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油料树种千年桐的SSR标记开发、遗传多样性与群体结构分析

赵盼, 栗丹阳, 马锦林, 梁文汇, 庞晓明, 龙萃, 马婧怡, 郭惠红

赵盼, 栗丹阳, 马锦林, 梁文汇, 庞晓明, 龙萃, 马婧怡, 郭惠红. 油料树种千年桐的SSR标记开发、遗传多样性与群体结构分析[J]. 北京林业大学学报, 2021, 43(11): 50-61. DOI: 10.12171/j.1000-1522.20210054
引用本文: 赵盼, 栗丹阳, 马锦林, 梁文汇, 庞晓明, 龙萃, 马婧怡, 郭惠红. 油料树种千年桐的SSR标记开发、遗传多样性与群体结构分析[J]. 北京林业大学学报, 2021, 43(11): 50-61. DOI: 10.12171/j.1000-1522.20210054
Zhao Pan, Li Danyang, Ma Jinlin, Liang Wenhui, Pang Xiaoming, Long Cui, Ma Jingyi, Guo Huihong. SSR marker development, genetic diversity and population structure analysis in oil tree species Vernicia montana[J]. Journal of Beijing Forestry University, 2021, 43(11): 50-61. DOI: 10.12171/j.1000-1522.20210054
Citation: Zhao Pan, Li Danyang, Ma Jinlin, Liang Wenhui, Pang Xiaoming, Long Cui, Ma Jingyi, Guo Huihong. SSR marker development, genetic diversity and population structure analysis in oil tree species Vernicia montana[J]. Journal of Beijing Forestry University, 2021, 43(11): 50-61. DOI: 10.12171/j.1000-1522.20210054

油料树种千年桐的SSR标记开发、遗传多样性与群体结构分析

基金项目: 中央高校基本科研业务费专项(2017ZY24),国家自然科学基金项目(31870650)
详细信息
    作者简介:

    赵盼。主要研究方向:树木生长发育及调控。Email:zhaopan11250528@163.com 地址:100083 北京市海淀区清华东路35号北京林业大学生物科学与技术学院

    责任作者:

    郭惠红,教授。主要研究方向:树木生长发育及调控。Email:guohh@bjfu.edu.cn 地址:同上

  • 中图分类号: S792.43

SSR marker development, genetic diversity and population structure analysis in oil tree species Vernicia montana

  • 摘要:
      目的  千年桐是大戟科油桐属的一种重要的工业油料树种,较同属的油桐具有更强的抗枯萎病能力,近年来受到了广泛关注。然而,因千年桐栽培历史短及种质管理的不足,目前对其分子遗传方面的研究还非常有限。本研究旨在开发千年桐的基因组SSR标记,进而开展其种质的鉴定、遗传多样性与群体结构分析。
      方法  采用RAD测序技术获得千年桐简化基因组以开发SSR标记,基于SSR标记利用分子变异分析(AMOVA)、非加权组平均法(UPGMA)聚类、主坐标(PCoA)与群体结构分析等方法对来自3个不同地理分布的共105份千年桐种质资源进行研究。
      结果  17个多态性的三核苷酸基因组SSR标记被开发,并能够很好地鉴别所有收集的105份千年桐种质。在其中62份种质中检测到85个私有等位基因,涉及15个SSR位点。AMOVA分析发现,千年桐群体间呈现出中等程度的遗传分化,但群体内的遗传变异远高于群体间的遗传变异。群体结构分析显示,3个来自不同地理分布的千年桐群体中存在4个不同的基因库,群体间既有进化独立性,又有较高程度的遗传混合,这一结果与UPGMA和PCoA分析的结果基本一致。
      结论  新开发的17个SSR标记有效鉴定了105份千年桐种质,揭示了其遗传多样性和群体遗传结构,对千年桐种质保存和育种计划具有非常重要的参考价值。
    Abstract:
      Objective  Vernicia montana, belonging to the genus Vernicia of Euphorbiaceae family, is an important industrial oil tree species, which has received great attention in recent years because it is much more resistant to wilt disease than V. fordii of same genus. However, the current researches on the molecular genetics of V. montana are still very limited due to its short cultivation history and inadequate management of germplasm resources. This study aimed to develop V. montana’s genome SSR markers, and then to carry out its germplasm identification, genetic diversity and population structure analyses.
      Method  Using RAD sequencing technology to obtain a simplified genome of V. montana to develop its SSR markers, based on the SSR markers, a series of methods including the analysis of molecular variance (AMOVA), the unweighted pair group method with arithmetic mean (UPGMA) clustering, principal coordinate analysis (PCoA) and population structure analysis were used to study 105 germplasm resources of V. montana from three different geographical distributions.
      Result  17 polymorphic trinucleotide genomic-SSR markers were developed, which well distinguished all the tested 105 V. montana germplasms. A total of 85 private alleles were detected in 62 germplasms at 15 SSR loci. The AMOVA analysis revealed a moderate degree of genetic differentiation among the populations of V. montana; however, the genetic variation within the populations was much higher than that among the populations. Population structure analysis showed that four different gene pools were present in the three V. montana populations from different geographical distributions, and there was both evolutionary independence and a relatively high degree of genetic admixture among the populations, which was basically consistent with the results of UPGMA and PCoA analyses.
      Conclusion  The newly developed 17 SSR markers effectively identified 105 V. montana germplasms, and revealed the genetic diversity and population genetic structure of the germplasms, which would be very helpful for the conservation of germplasms and breeding program in V. montana.
  • 油桐(Vernicia fordii)和千年桐(V. montana)是大戟科(Euphorbiaceae)油桐属(Vernicia)中两个重要的工业木本油料树种,主要分布在中国南部和一些东南亚国家[1-2]。这两个树种的种子油富含共轭三烯十八碳不饱和脂肪酸即α-硬脂酸,是著名的工业干燥油[3],已被广泛用于油漆、油墨等工业用料和生物柴油的生产[2, 4-5]。与油桐相比,千年桐的栽培和研究历史相对较短,但有研究表明其种子油产量高于油桐[1]。此外,千年桐对于土壤传播的镰刀菌枯萎病的抵抗力要显著强于油桐。在栽培中发现,枯萎病会严重阻碍油桐的生长与发育,而以千年桐作砧木,将油桐嫁接到千年桐上可以很好地抵抗枯萎病[6]。这些研究表明,千年桐具有更好的开发和利用价值。然而,千年桐栽培和发展历史较短,导致对其种质的管理不完善[1, 7],相关种质鉴定和遗传多样性的研究还未充分开展。因此,本研究开展千年桐的种质鉴定和遗传多样性分析对于其种质保存、高产和抗病的新品种培育具有重要的实践意义。

    近年来,分子标记已被证明是鉴定植物种质和揭示种质遗传多样性的有效工具[8-9]。许多类型的分子标记已被开发并广泛使用,包括限制性酶切片段长度多态性标记(restriction fragment length polymorphism,RFLP),扩增片段长度多态性标记(amplified fragment length polymorphism,AFLP),简单重复序列标记(simple sequence repeat,SSR),简单重复序列区间标记(inter-simple sequence repeat,ISSR)和单核苷酸多态性标记(single nucleotide polymorphism,SNP)[8, 10-11]。其中,SSR标记由于在基因组中的丰富性、高多态性、可再现性和共显性遗传等特点,已成为最优选的分子标记类型之一[12-13],已成功应用于油桐、油茶(Camellia oleifera)和油橄榄(Olea europaea)等多种木本油料树种的种质鉴定、遗传多样性和群体结构分析等研究[14-18]。然而,SSR标记的应用在千年桐中非常有限。迄今为止,仅一篇关于千年桐SSR标记开发和应用的研究报道,且其开发的基因组SSR标记仅基于二核苷酸重复序列[3]。与较长的SSR重复序列类型相比,二核苷酸SSR标记在识别大小接近的等位基因时,不易检测到其差异,从而容易导致错误的判断或产生混淆[19-20]。因此,具有较长重复核心的SSR标记将更有利于千年桐的种质鉴定和遗传分析。

    针对上述问题,本研究基于限制性位点关联DNA(restriction-site associated DNA,RAD)测序技术开发多态性的三核苷酸基因组SSR标记,对广西省收集的105份千年桐种质资源进行基因分型。开发的SSR标记在群体水平上进一步研究105份千年桐种质资源的遗传多样性和结构。

    本研究从千年桐种质资源最丰富的中国广西壮族自治区收集到105份千年桐种质资源。根据其地理位置的分布情况,将这105份千年桐种质资源划分为3个群体。其中群体1分布于河池市凤山县(27份),群体2分布于百色市田林县(34份),群体3分布于南宁市西乡塘区(44份)。其中群体3中的“桂皱1号”“桂皱2号”“桂皱6号”和“桂皱27号”4个高产优良无性系是由广西林业科学研究院选育出的优良品种[21]。为了选择具有代表性的种质,所收集的千年桐种质资源之间的间隔至少为100 m,每一份种质由一棵树代表。

    利用植物DNA提取试剂盒(DP320-03,天根生化科技有限公司,北京)提取105份千年桐样品的总DNA,经质检合格后基于RAD测序技术构建测序文库,在Illumina平台进行高通量测序(ORI-GENE,中国北京)。高通量测序得到的原始数据(raw reads)过滤掉包含接头、poly N和poly A等低质量的序列后得到高质量的过滤后数据(cleaned reads),使用Trinity和FLASH软件将过滤后的短序列拼接得到重叠群(contigs),然后进一步组装成简化的基因组。

    使用基于Perl脚本的MIcroSAtellite鉴定工具(MISA)扫描千年桐RAD测序文库以鉴别SSR位点,扫描的标准是三核苷酸基序至少5个重复,四和五核苷酸基序至少4个重复。SSR引物通过Primer Premier 5软件设计,由睿博兴科生物公司合成。PCR反应体系的总体积为20 μL,其中包含40 ~ 60 ng基因组DNA,25 μmol/L的dNTP,2.5 U的Taq DNA聚合酶,10 μmol/L的正、反向引物,10 μmol/L的荧光染料,及含有25 mmol/L Mg2+的10 × PCR缓冲液(天根生化科技有限公司,北京)。PCR反应程序采用两步法:94 ℃预变性5 min,然后94 ℃变性30 s,55 ℃退火40 s,72 ℃延伸30 s,共30个循环,接着进行94 ℃变性30 s,53 ℃退火40 s,72 ℃延伸30 s,共8个循环,最后72 ℃完全反应10 min。通过毛细管电泳检测具有荧光标记的PCR产物。每个试验进行3次重复。

    通过GeneMarker v2.2.0软件分析从毛细管电泳获得的SSR原始数据。使用POPGENE 32和Cervus v3.0.7软件计算每个SSR位点的遗传多样性信息参数,包括观察到的等位基因(Na)、有效等位基因(Ne),观察到的杂合度(Ho)、预期的杂合度(He)、香农信息指数(I)、基因流(Nm)、遗传分化系数(FST)、无效等位基因频率(FNull)和多态性信息含量(PIC)。使用GenALEx v6.503软件进行哈迪−温伯格平衡检测(HWE),同时检测105份千年桐种质资源中的私有等位基因。

    GenALEx v6.503软件也用于AMOVA分子变异分析,同时AMOVA中的FST用于评估群体遗传分化。使用PowerMarker v3.25软件计算Nei’s遗传距离[22],基于遗传距离,使用非加权组平均法(UPGMA)对105份千年桐种质进行聚类分析。为了获得更直观的遗传分类,利用iTOL在线工具进一步注释系统发育树。同时,在GenALEx v6.6.503软件中使用上述遗传距离数据矩阵进行主坐标分析(PCoA)。使用STRUCTURE v2.3软件对105份千年桐种质资源进行群体遗传结构分析。在标准STRUCTURE分析中,假定的种群数量用K值表示,设置其范围为1 ~ 8,对8个K值进行逐一测试,并针对每个K值执行5次独立运行。通过Structure Harvester程序使用Delta−K法评估最佳种群数量的K值。

    本研究运用RAD测序技术共得到45.932 Mbp的原始数据,去除低质量的序列后获得30.996 Mbp的过滤后数据,占原始数据的67.5%(表1)。Q20和Q30分别为98.3%和95.4%,GC含量为40.51%(表1),这表明测序质量很高,数据可靠。过滤后的短序列经过拼接获得了139 988条重叠群,其平均长度为256 bp(表1),然后进一步组装成简化基因组。在千年桐简化基因组中共鉴定到3 425个SSR位点(表2),其中包括2 174个二核苷酸SSR位点,1 023个三核苷酸SSR位点,131个四核苷酸SSR位点,及97个五核苷酸SSR位点,分别占总SSR位点的63.47%、29.87%、3.83%和2.83%。除二核苷酸SSR位点外,剩余1 251个三、四、五核苷酸SSR位点均被用于基因组SSR标记的开发。

    表  1  测序数据质量统计总表
    Table  1.  Quality summary of sequencing data
    原始数据
    Raw read/Mbp
    原始Q20
    Raw Q20/Gbp
    原始Q30
    Raw Q30/Gbp
    过滤后数据
    Cleaned read/Mbp
    过滤后Q20
    Cleaned Q20/Gbp
    过滤后Q30
    Cleaned Q30/Gbp
    平均长度
    Average length/bp
    GC含量
    GC content/%
    45.9326.580 (95.5%)6.257 (90.8%)30.996 (67.5%)4.510 (98.3%)4.374 (95.4%)148.040.51
    注:Q20. 质量值 ≥ 20的碱基所占百分比;Q30. 质量值 ≥ 30的碱基所占百分比。Notes: Q20, percentage of bases with the quanlity value ≥ 20; Q30, percentage of bases with the quanlity value ≥ 30.
    下载: 导出CSV 
    | 显示表格
    表  2  具有不同重复基序的SSR位点在千年桐基因组中的分布
    Table  2.  Distribution of SSR with different repeat motifs in the genome of Vernicia montana
    SSR重复基序
    SSR repeat motif
    SSR数量
    Number of SSR
    频率
    Frequency/%
    二核苷酸 Dinucleotide 2 174 63.47
    三核苷酸 Trinucleotide 1 023 29.87
    四核苷酸 Tetranucleotide 131 3.83
    五核苷酸 Pentanucleotide 97 2.83
    总计 Total 3 425 100.00
    下载: 导出CSV 
    | 显示表格

    针对上述1 251个三、四、五核苷酸SSR位点,共设计得到300对SSR引物,其中包括250对三核苷酸SSR引物、28对四核苷酸SSR引物和22对五核苷酸SSR引物。利用这300对引物对随机选取的一份千年桐种质资源(“桂皱1号”)的基因组DNA进行PCR扩增,毛细管电泳检测发现其中173对SSR引物扩增出了预期片段大小的PCR产物,具体包括162对、5对和6对三核苷酸、四核苷酸和五核苷酸SSR引物。这173对SSR引物被进一步在8份随机选取的千年桐种质资源中进行基因分型,筛选出56对多态性引物,包括52对三核苷酸SSR引物和4对五核苷酸SSR引物。随后,56对SSR引物被用于所有105份千年桐种质资源的基因分型,最终获得了17个能够完全鉴别105份千年桐种质资源的多态性SSR标记(表3)。

    表  3  针对105份千年桐种质开发的17个多态性SSR标记的信息
    Table  3.  Information of 17 polymorphic SSR markers developed for 105 V. montana germplasms
    引物编号
    Primer No.
    重复基序
    Repeat motif
    引物序列(5′—3′)
    Primer sequence (5′−3′)
    退火温度
    Annealing temperature/℃
    预期大小
    Expected size/bp
    Vm-BFU01 (TTG)5 AACCACTGCTACTTCACCATTTTC 55 167
    ACCCAATGTTTTCTCCGACC
    Vm-BFU02 (AGA)5 GTGTTGAGCCAGAAACCCATTA 55 165
    CAGAGAAGCCTCGGTCCCTA
    Vm-BFU03 (CAT)6 TCTACTCCCACACTTCCAAAACA 55 161
    TAATCTCCTTCTGTCCTTCACGA
    Vm-BFU04 (CAG)9 GGGAGTGGTGGCAATGGC 55 181
    GCTGGGAGGCATTGTTGAAG
    Vm-BFU05 (AGA)9 GAGCCAAGAGAAGACGAAAAGAG 55 146
    ACCGTTTACAGTGTTTCGCTATG
    Vm-BFU06 (GGA)8 TGTGCCGCTTGTAACTGCC 55 164
    TGCGGCTGTGTCAGGTGTAG
    Vm-BFU07 (TCT)7 AGCCTTTGCCACTGTTGAGC 55 127
    GATGGGTCCGCCAAGTTCA
    Vm-BFU08 (TCT)5 ATTGTGAAGGATTTGCGATGG 55 161
    CGGCGAAAACGAAACAGAG
    Vm-BFU09 (GTT)7 TCGCCTAAGGTGGTCTTGATG 55 178
    GCCCAACGAAATCTAACTCTAATAA
    Vm-BFU10 (TAA)12 TTCCTCCTCTGGTGACGCTT 55 252
    TTCCTTCCATCATCAACTTTTACC
    Vm-BFU11 (TCC)7 GCCGCCGCCTACTACTTACTT 55 131
    TTTCTCAAACCAAACAGGAGTTG
    Vm-BFU12 (CTT)5 TATTTTTCTTGGGAGTAAAGTCACC 55 198
    TATGTGAAATGGAGAGTTCGGAG
    Vm-BFU13 (CAG)6 TTGTCAACAAGCCTTCTCACCT 55 160
    GCTCCAAGTCCCATCATCATTT
    Vm-BFU14 (GGC)5 CCAAAACCATCAATCTCTTCGC 55 204
    TGATTTCGCACAAGTCCCAAG
    Vm-BFU15 (ACC)10 GCGTTCCTGACCCTACCTTT 55 184
    AGAGAAACAAAAAAGCCACCAG
    Vm-BFU16 (TTA)12 TCCCCACAGCCATAAAACAAG 55 199
    TTTCCAAAACTCTCAAACCACAA
    Vm-BFU17 (AAT)10 ACCCCATCTATGACATCCCACT 55 195
    CCCCGTTCTTGCTCTCCC
    注:重复基序括号外的数字为其重复次数。Note: the number of repeats is marked outside the parentheses of repeat motifs.
    下载: 导出CSV 
    | 显示表格

    对于所开发的17个SSR标记,PIC的值在0.309 ~ 0.824之间,平均值为0.552,其中10个为高度多态性标记(PIC ≥ 0.50)、7个为中度多态性标记(0.25 ≤ PIC < 0.50)。基于17个SSR标记,在105份千年桐种质中共检测到138个等位基因,每个SSR位点Na最少为2个,最多达14个,平均为8.117个(表4)。值得注意的是,遗传多样性评价的结果显示了私有等位基因(number of private allele,NPA)的存在。在17个多态性SSR位点中的15个位点上共检测到85个私有等位基因,平均每个位点有5个私有等位基因(表4表5)。这85个私有等位基因存在于62份千年桐种质中,其中7份种质来自于群体1,22份种质来自于群体2,33份种质自于群体3(表5)。此外,其他遗传多样性评价参数,如HoI及Nm等与PIC呈现正相关的关系(表4)。

    表  4  17个千年桐SSR位点的遗传多样性参数
    Table  4.  Genetic diversity parameters of 17 SSR loci in V. montana
    位点 LocusNaNeHoHeIFNullPICFSTNmHWENPA
    Vm-BFU012.0001.6180.1900.3840.5700.3350.3090.1471.446***0
    Vm-BFU026.0001.4910.0860.3310.7040.5900.3100.01220.486***1
    Vm-BFU0310.0002.4000.3650.5861.2230.2180.5390.0554.286***9
    Vm-BFU0411.0003.2620.4760.6971.6200.1780.6700.0643.629***3
    Vm-BFU054.0002.1870.2860.5460.9230.3060.4660.01813.956***1
    Vm-BFU065.0002.5950.4170.6181.0750.1790.5470.01813.645***1
    Vm-BFU076.0003.1350.4190.6841.3570.2320.6380.0356.850***2
    Vm-BFU088.0002.1910.5830.5461.1600.0540.5100.0347.124***2
    Vm-BFU094.0002.2370.3900.5560.9080.1620.4530.0723.214***6
    Vm-BFU109.0002.0780.2000.5211.1490.4500.4910.0633.695***12
    Vm-BFU1113.0006.2890.2350.8452.1010.5590.8240.0574.163***3
    Vm-BFU122.0001.8730.3200.4690.6590.1860.3580.0504.742**0
    Vm-BFU1314.0003.1570.4100.6871.6170.2520.6570.0932.439***10
    Vm-BFU1410.0001.8860.2480.4721.1440.3040.4570.0972.338***15
    Vm-BFU1511.0003.1550.3520.6861.5440.3330.6480.0623.808***10
    Vm-BFU169.0005.2530.5480.8141.7870.1930.7820.0564.256***2
    Vm-BFU1714.0003.9130.5380.7481.8520.1580.7250.0317.705***8
    总数 Total138.00048.72885
    平均值 Mean8.1172.8660.3570.5991.2580.2760.5520.0564.2515
    注:Na. 观测到的等位基因数;Ne. 有效等位基因数;Ho. 观测到的杂合度;He. 预期杂合度;I. 香农信息指数;Nm. 基因流;FNull. 无效等位基因频率;PIC. 多态信息含量;FST. 遗传分化指数;HWE. 哈迪−温伯格平衡;*. P < 0.05;**. P < 0.01; ***. P < 0.001;NPA. 私有等位基因的数量。下同。Notes: Na, observed number of alleles; Ne, effective number of alleles; Ho, observed heterozygosity; He, expected heterozygosity; I, Shannon’s information index; Nm, gene flow; FNull, null allele frequency; PIC, polymorphism information content; FST, genetic differentiation index; HWE, Hardy-Weinberg equilibrium; * means P < 0.05; ** means P < 0.01; *** means P < 0.001; NPA, number of private alleles. The same below.
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    表  5  具有一至多个私有等位基因的千年桐种质
    Table  5.  V. montana germplasms with one or more private alleles
    种质
    Germplasm
    群体
    Population
    NPA私有等位基因位点
    Locus of private allele
    种质
    Germplasm
    群体
    Population
    NPA私有等位基因位点
    Locus of private allele
    1-3 1 1 Vm-BFU11 3-3 3 2 Vm-BFU03, Vm-BFU17
    1-4 1 2 Vm-BFU03, Vm-BFU07 3-4 3 3 Vm-BFU03, Vm-BFU14, Vm-BFU16
    1-7 1 2 Vm-BFU03, Vm-BFU07 3-5 3 1 Vm-BFU14
    1-15 1 1 Vm-BFU06 3-6 3 1 Vm-BFU13
    1-18 1 1 Vm-BFU11 3-9 3 3 Vm-BFU03, Vm-BFU11, Vm-BFU13
    1-23 1 1 Vm-BFU13 3-10 3 1 Vm-BFU15
    1-27 1 2 Vm-BFU10, Vm-BFU17 3-12 3 1 Vm-BFU14
    2-2 2 1 Vm-BFU10 3-13 3 1 Vm-BFU15
    2-3 2 1 Vm-BFU09 3-14 3 2 Vm-BFU14, Vm-BFU17
    2-4 2 1 Vm-BFU10 3-15 3 1 Vm-BFU13
    2-5 2 3 Vm-BFU09, Vm-BFU10, Vm-BFU13 3-16 3 1 Vm-BFU14
    2-6 2 2 Vm-BFU03, Vm-BFU13 3-17 3 1 Vm-BFU14
    2-11 2 1 Vm-BFU10 3-22 3 1 Vm-BFU03
    2-13 2 1 Vm-BFU15 3-23 3 2 Vm-BFU04, Vm-BFU14
    2-14 2 2 Vm-BFU10, Vm-BFU15 3-24 3 1 Vm-BFU15
    2-15 2 1 Vm-BFU09 3-25 3 3 Vm-BFU04, Vm-BFU14, Vm-BFU15
    2-16 2 1 Vm-BFU09 3-26 3 1 Vm-BFU14
    2-17 2 1 Vm-BFU09 3-27 3 2 Vm-BFU15, Vm-BFU17
    2-19 2 1 Vm-BFU15 3-28 3 2 Vm-BFU08, Vm-BFU17
    2-20 2 1 Vm-BFU10 3-29 3 1 Vm-BFU14
    2-23 2 1 Vm-BFU10 3-30 3 1 Vm-BFU14
    2-24 2 1 Vm-BFU09 3-31 3 1 Vm-BFU14
    2-26 2 1 Vm-BFU02 3-32 3 2 Vm-BFU03, Vm-BFU05
    2-27 2 2 Vm-BFU10, Vm-BFU15 3-33 3 1 Vm-BFU16
    2-28 2 1 Vm-BFU13 3-35 3 1 Vm-BFU14
    2-29 2 1 Vm-BFU13 10-7 3 1 Vm-BFU17
    2-30 2 2 Vm-BFU10, Vm-BFU13 10-13 3 2 Vm-BFU03, Vm-BFU15
    2-31 2 1 Vm-BFU13 10-16 3 1 Vm-BFU14
    2-33 2 1 Vm-BFU10 10-18 3 1 Vm-BFU10
    3-1 3 1 Vm-BFU17 Guizhou-2 3 2 Vm-BFU08, Vm-BFU17
    3-2 3 1 Vm-BFU14 Guizhou-27 3 1 Vm-BFU04
    总计 Total 85
    下载: 导出CSV 
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    遗传分化指数(FST)是评估群体间遗传分化程度的参数。本研究利用POPGENE软件分析得出:17个SSR标记的FST的范围在0.012 ~ 0.147之间,平均值为0.056(表4)。利用GenALEx软件计算得出的FST值为0.080(P < 0.001)(表6),与POPGENE的结果基本一致。基于所开发的17个SSR标记,AMOVA分子变异分析进一步显示,千年桐群体内的遗传变异远高于群体间的遗传变异,其群体内与群体间的遗传变异分别占总变异的92%和8%。

    表  6  千年桐群体间与群体内的AMOVA分析
    Table  6.  AMOVA analysis among and within V. montana populations
    变异来源
    Source of variation
    自由度
    Degree of freedom
    方差和
    Sum of squares
    变异组分
    Variance component
    占总变异的比例
    Proportion in total variation/%
    FST
    群体间 Among populations 2 110.841 1.209 8 0.080 (P < 0.001)
    群体内 Within population 102 1 423.025 13.951 92
    总计 Total 104 1 533.867 15.160 100
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    基于SSR位点的哈迪−温伯格平衡能够评估群体是否存在连锁不平衡,判断是否偏离遗传平衡,具体用显著性水平P−value来表示。当P > 0.05时,群体符合遗传平衡;而当P < 0.05时,群体显著偏离遗传平衡[23]。本研究中,17个多态性SSR位点经Bonferroni校正后显示,千年桐群体全部显著偏离哈迪−温伯格平衡(P < 0.05)(表4)。

    基于Nei’s遗传距离、利用UPGMA方法对来自3个群体的105份千年桐种质进行聚类分析,结果显示(图1):群体1、2和3的部分种质(约占全部种质的三分之一)分别单独聚集在Ⅰ、Ⅱ和Ⅲ这3个分支中;来自3个群体的部分种质(约占全部种质的三分之一)共同聚集在较大的分支Ⅳ中;来自群体1和3的部分种质(不到种质总数的三分之一)共同聚集在分支Ⅴ中。

    图  1  基于17个SSR标记的105份千年桐种质资源遗传关系的进化树
    红色、黄色和蓝色的线段分别代表群体1、2和3。The red, yellow and blue lines represent the population 1, 2 and 3, respectively.
    Figure  1.  A dendrogram of genetic relationship among 105 V. montana germplasm resources based on 17 SSR markers

    105份千年桐种质的PCoA分析结果显示,第一个主坐标和第二个主坐标分别占所有千年桐种质总遗传变异的14.07%和9.01%(图2)。PCoA分析结果与UPGMA聚类分析的结果基本一致。

    图  2  基于17个SSR标记的105份千年桐种质资源的主坐标分析(PCoA)
    红色、黄色和蓝色的点分别代表群体1、2和3。The red, yellow and blue dots represent population 1, 2 and 3, respectively.
    Figure  2.  Principal coordinate analysis (PCoA) of the 105 V. montana germplsm resources based on 17 SSR markers

    为了揭示105份千年桐种质的群体结构,基于贝叶斯的方法分析了来自不同地理分布的3个群体的遗传结构,其中推定的种群数量(基因库)用K值表示。利用标准STRUCTURE分析,对8个K值(K = 1 ~ 8)逐一测试,根据Delta K值确定了最佳种群数量为2(K = 2),其次为4(K = 4)(图3ab)。考虑到标准STRUCTURE方法中软件可能存在的偏差,因此分别在K = 2和K = 4的情况下分析了来自3个群体的105份千年桐种质的遗传结构(图3cd)。结果表明,K = 4的群体结构比K = 2的群体结构更合理,因为来自不同地理分布的3个群体的所有种质都以各自独特的基因库出现在K = 4而不是K = 2的群体结构中。如图3所示,来自不同地理分布的3个千年桐群体中存在4个基因库。群体1、2和3分别由红色、黄色和蓝色片段代表的基因库主导,而绿色片段代表的基因库散布在这3个群体中,其在群体2和群体3中出现的频率比在群体1中更高。因此,在这105份千年桐种质资源中,基于群体结构推定的种群接近于地理划分的种群。

    图  3  基于17个SSR标记的105份千年桐种质资源的群体结构分析
    a. K = 1 ~ 8时Delta K的变化图;b. 基于每个K值的5个重复,使用ln P(D)的平均值进行种群估计;c. 105份千年桐种质在K = 2时的种群结构;d. 105份千年桐种质在K = 4时的种群结构。a, plot of Delta K with K ranging from 1 to 8; b, estimation of population using the mean of ln P(D) based on five repetitions for each K-value; c, population structure of 105 V. montana germplasms at K = 2; d, population structure of 105 V. montana germplasms at K = 4.
    Figure  3.  Population structure analysis of 105 V. montana germplasm resources based on 17 SSR markers

    RAD测序是一种基于全基因组酶切位点的简化基因组测序技术。它可以大规模快速鉴定基因组中的SSR位点,并具有高通量、高精度、低成本和短周期的优势[24-25]。对于没有参考基因组的物种,RAD测序技术是一种开发基因组SSR标记的替代方法。近年来,通过RAD测序技术已在大麦(Hordeum vulgare)、甘薯(Dioscorea esculenta)等多种植物中成功开发了SSR标记[25-27],证实了RAD测序技术的可行性。因此,本研究首次利用RAD测序技术获得了无参考基因组的千年桐的简化基因组,开发SSR标记来鉴定种质,并分析千年桐种质的遗传多样性和群体结构。

    SSR标记是植物基因组中以1 ~ 6个碱基为核心单位组成的串联重复DNA序列[28]。与单核苷酸和双核苷酸SSR标记相比,具有较长重复核心的三核苷酸、四核苷酸和五核苷酸SSR标记更容易检测到等位基因长度上的差异,因而更利于种质的鉴别[28-29]。在以往的千年桐研究中,只有一项研究开发了20对二核苷酸SSR标记来鉴别30份千年桐种质[3]。在本研究中,通过对300个包括三、四和五核苷酸SSR标记的筛选,最终发现只有17个多态性的三核苷酸SSR标记可以很好地鉴别所有105份千年桐种质(表2表3),说明三核苷酸SSR标记对于鉴别千年桐种质是最有效的。三核苷酸SSR标记在种质鉴定中的高效性也已在其他植物中得到证实,例如大豆(Glycine max)、猕猴桃(Actinidia chinensis)、鹰嘴豆(Cicer arietinum)和油橄榄[18, 30-32]

    PIC作为一种重要的遗传多样性参数,代表了SSR位点的变异程度,可以用来评估SSR标记的鉴别能力,且不受种质数量、地理起源和所调查的基因位点的影响[33-34]。在本研究中,所开发的17个基因组SSR标记的平均PIC值为0.552(表3),高于油桐中开发的22个EST-SSR标记的平均PIC值(0.401)[1]。基因组SSR标记的多态性高于由转录组开发的EST-SSR标记的多态性,这在核桃(Juglans regia)的研究中也有报道[35-36],这种现象很可能是由于基因组中含有不保守的内含子所致。

    本研究所开发的基因组SSR位点,除Vm-BFU08外,观察到的杂合度(Ho)均低于预期的杂合度(He)(表4)。在核桃和白柳(Salix alba)中也观察到了类似的现象[37-38],这被认为是由于纯合子过多或无效等位基因存在所致[37, 39]。在千年桐的一些SSR位点中,如Vm-BFU01、Vm-BFU02,Vm-BFU05、Vm-BFU10和Vm-BFU15,出现了过多的纯合子,而Vm-BFU11和Vm-BFU16位点显示出较高的无效等位基因频率(表4)。因此,本研究中SSR位点Ho低于He也是由过多纯合子和无效等位基因两方面原因导致。对于无效等位基因的存在,被认为是引物结合位点上插入或缺失突变导致的SSR位点侧翼序列变异引起的[40-41]

    私有等位基因的存在可以在一定程度上反映植物种质资源的遗传多样性[42-43],这对种质的鉴定和后续的育种工作具有重要的指导意义。在本研究中,占总种质一半以上的千年桐种质资源(62份)中发现含私有等位基因(表4表5),这揭示了105份千年桐种质资源中存在相对广泛的遗传变异。在植物栽培和驯化过程中,私有等位基因的存在被认为是由基因交换或可能的突变引起的[18, 44]。值得注意的是,在这62份具有私有等位基因的种质资源中,有一半以上来自群体3(表5),这表明群体3的种质比群体1和2具有更广泛的遗传变异。

    遗传分化指数(FST)可以评估群体间遗传分化的程度[44]FST < 0.05、0.05 < FST < 0.15、0.15 < FST < 0.25和FST > 0.25分别表示弱、中、高和极高的群体遗传分化水平[45]。在本研究中,由POPGENE和AMOVA分析计算的FST值在0.012至0.147之间(表4表6),表明所测千年桐群体间存在中度程度的遗传分化。然而,千年桐群体内的遗传变异远高于群体间(表6),这一现象在多种木本植物中也被发现,例如核桃[35]、油棕(Elaeis guineensis[46]、白柳[38]、油桐[7]和杉木(Cunninghamia lanceolata[13]。造成这种现象的原因可能与不同群体种质资源之间的基因交流频率增加有关[13, 46]。在群体遗传学中,通常采用哈迪−温伯格平衡(HWE)来检测群体是否存在连锁不平衡和群体遗传混合的程度[47]。在本研究中,基于17个SSR位点的3个千年桐群体明显偏离了哈迪−温伯格平衡(表4)。一般说来,群体偏离哈迪−温伯格平衡被认为可能是SSR位点的杂合度或纯合度过高所致[38]。在本研究中发现,17个SSR位点中的大部分位点的杂合度过高(表4),这可能是千年桐群体偏离哈迪−温伯格平衡的主要原因,同时也反映了一定程度的群体遗传混合。在自然条件下,隔离、选择、杂交和漂移等过程会影响群体遗传结构的形成,而这些过程也会受到人为干预的影响[48],从而可能导致SSR位点的杂合度过高。

    群体结构分析显示,来自不同地理分布的3个千年桐群体既有进化上的相对独立性又有较高程度的遗传混合(图3),这与UPGMA和PCoA的结果基本一致(图1图2)。植物群体之间的遗传混合通常与地理位置或地理环境密切相关[38, 44]。在土耳其的2个河流生态系统中,广泛人为介导的植物材料迁移显著影响了白柳群体的遗传结构[38]。在本研究中,3个群体的千年桐种质资源分别采自广西省中部红水河流域自西向东的3个不同地点。红水河流域具有丰富的水资源和植被资源,且该河流生态系统中人类活动较为普遍[49-50]。因此,千年桐3个群体之间较高程度的遗传混合与红水河流域及周边地区的人类活动密切相关。在一些天然河流生态系统中,河流下游的植物群体的遗传多样性高于上游,比如当归(Angelica archangelica)、疏花水柏枝(Myricaria laxiflora)和柳树(Salix hukaoana[51-53]。植物种子或营养繁殖体通过河流系统自上游向下游流动可以解释这种现象,也能解释红水河下游的群体3比红水河上游的群体1和2遗传混合高的现象(图1 ~ 3)。群体3中含私有等位基因的种质资源也最多,与上述现象有较好的相关性。而对于红水河上游的2个群体,群体2的遗传混合高于群体1(图3),这很可能是受到了人类活动的干扰。人类活动对植物种质资源遗传混合的影响常见于一些国内外分布较广的植物物种中,包括油茶[15]、椰子(Cocos nucifera[54]、核桃[16, 35-36]、油棕[17]和油桐[7]。例如,与千年桐同属的油桐,人类携带其种子在产地间迁移等活动对其种质资源的遗传混合产生了显著影响[7]。值得注意的是,本研究中利用群体结构分析推定的群体数量(基因库)略多于从不同地理起源收集的群体数量(图3),类似的现象也在葡萄(Vitis vinifera)、油橄榄和石榴(Punica granatum)的群体遗传结构分析中被发现[9, 41, 55]。因此,本研究中的3个千年桐群体可能由于历史起源确实存在4个基因库,但也不排除其由于群体间的基因交换、基因突变或其他原因创建了1个新的基因库。

    综上所述,本研究首次在千年桐中成功开发了17个多态性的三核苷酸基因组SSR标记。这17个SSR标记能很好地鉴别来自不同地理分布的3个千年桐群体的105份种质资源,并揭示了种质的遗传多样性和群体遗传结构,为千年桐的种质保存和育种提供了有价值的科学依据。

  • 图  1   基于17个SSR标记的105份千年桐种质资源遗传关系的进化树

    红色、黄色和蓝色的线段分别代表群体1、2和3。The red, yellow and blue lines represent the population 1, 2 and 3, respectively.

    Figure  1.   A dendrogram of genetic relationship among 105 V. montana germplasm resources based on 17 SSR markers

    图  2   基于17个SSR标记的105份千年桐种质资源的主坐标分析(PCoA)

    红色、黄色和蓝色的点分别代表群体1、2和3。The red, yellow and blue dots represent population 1, 2 and 3, respectively.

    Figure  2.   Principal coordinate analysis (PCoA) of the 105 V. montana germplsm resources based on 17 SSR markers

    图  3   基于17个SSR标记的105份千年桐种质资源的群体结构分析

    a. K = 1 ~ 8时Delta K的变化图;b. 基于每个K值的5个重复,使用ln P(D)的平均值进行种群估计;c. 105份千年桐种质在K = 2时的种群结构;d. 105份千年桐种质在K = 4时的种群结构。a, plot of Delta K with K ranging from 1 to 8; b, estimation of population using the mean of ln P(D) based on five repetitions for each K-value; c, population structure of 105 V. montana germplasms at K = 2; d, population structure of 105 V. montana germplasms at K = 4.

    Figure  3.   Population structure analysis of 105 V. montana germplasm resources based on 17 SSR markers

    表  1   测序数据质量统计总表

    Table  1   Quality summary of sequencing data

    原始数据
    Raw read/Mbp
    原始Q20
    Raw Q20/Gbp
    原始Q30
    Raw Q30/Gbp
    过滤后数据
    Cleaned read/Mbp
    过滤后Q20
    Cleaned Q20/Gbp
    过滤后Q30
    Cleaned Q30/Gbp
    平均长度
    Average length/bp
    GC含量
    GC content/%
    45.9326.580 (95.5%)6.257 (90.8%)30.996 (67.5%)4.510 (98.3%)4.374 (95.4%)148.040.51
    注:Q20. 质量值 ≥ 20的碱基所占百分比;Q30. 质量值 ≥ 30的碱基所占百分比。Notes: Q20, percentage of bases with the quanlity value ≥ 20; Q30, percentage of bases with the quanlity value ≥ 30.
    下载: 导出CSV

    表  2   具有不同重复基序的SSR位点在千年桐基因组中的分布

    Table  2   Distribution of SSR with different repeat motifs in the genome of Vernicia montana

    SSR重复基序
    SSR repeat motif
    SSR数量
    Number of SSR
    频率
    Frequency/%
    二核苷酸 Dinucleotide 2 174 63.47
    三核苷酸 Trinucleotide 1 023 29.87
    四核苷酸 Tetranucleotide 131 3.83
    五核苷酸 Pentanucleotide 97 2.83
    总计 Total 3 425 100.00
    下载: 导出CSV

    表  3   针对105份千年桐种质开发的17个多态性SSR标记的信息

    Table  3   Information of 17 polymorphic SSR markers developed for 105 V. montana germplasms

    引物编号
    Primer No.
    重复基序
    Repeat motif
    引物序列(5′—3′)
    Primer sequence (5′−3′)
    退火温度
    Annealing temperature/℃
    预期大小
    Expected size/bp
    Vm-BFU01 (TTG)5 AACCACTGCTACTTCACCATTTTC 55 167
    ACCCAATGTTTTCTCCGACC
    Vm-BFU02 (AGA)5 GTGTTGAGCCAGAAACCCATTA 55 165
    CAGAGAAGCCTCGGTCCCTA
    Vm-BFU03 (CAT)6 TCTACTCCCACACTTCCAAAACA 55 161
    TAATCTCCTTCTGTCCTTCACGA
    Vm-BFU04 (CAG)9 GGGAGTGGTGGCAATGGC 55 181
    GCTGGGAGGCATTGTTGAAG
    Vm-BFU05 (AGA)9 GAGCCAAGAGAAGACGAAAAGAG 55 146
    ACCGTTTACAGTGTTTCGCTATG
    Vm-BFU06 (GGA)8 TGTGCCGCTTGTAACTGCC 55 164
    TGCGGCTGTGTCAGGTGTAG
    Vm-BFU07 (TCT)7 AGCCTTTGCCACTGTTGAGC 55 127
    GATGGGTCCGCCAAGTTCA
    Vm-BFU08 (TCT)5 ATTGTGAAGGATTTGCGATGG 55 161
    CGGCGAAAACGAAACAGAG
    Vm-BFU09 (GTT)7 TCGCCTAAGGTGGTCTTGATG 55 178
    GCCCAACGAAATCTAACTCTAATAA
    Vm-BFU10 (TAA)12 TTCCTCCTCTGGTGACGCTT 55 252
    TTCCTTCCATCATCAACTTTTACC
    Vm-BFU11 (TCC)7 GCCGCCGCCTACTACTTACTT 55 131
    TTTCTCAAACCAAACAGGAGTTG
    Vm-BFU12 (CTT)5 TATTTTTCTTGGGAGTAAAGTCACC 55 198
    TATGTGAAATGGAGAGTTCGGAG
    Vm-BFU13 (CAG)6 TTGTCAACAAGCCTTCTCACCT 55 160
    GCTCCAAGTCCCATCATCATTT
    Vm-BFU14 (GGC)5 CCAAAACCATCAATCTCTTCGC 55 204
    TGATTTCGCACAAGTCCCAAG
    Vm-BFU15 (ACC)10 GCGTTCCTGACCCTACCTTT 55 184
    AGAGAAACAAAAAAGCCACCAG
    Vm-BFU16 (TTA)12 TCCCCACAGCCATAAAACAAG 55 199
    TTTCCAAAACTCTCAAACCACAA
    Vm-BFU17 (AAT)10 ACCCCATCTATGACATCCCACT 55 195
    CCCCGTTCTTGCTCTCCC
    注:重复基序括号外的数字为其重复次数。Note: the number of repeats is marked outside the parentheses of repeat motifs.
    下载: 导出CSV

    表  4   17个千年桐SSR位点的遗传多样性参数

    Table  4   Genetic diversity parameters of 17 SSR loci in V. montana

    位点 LocusNaNeHoHeIFNullPICFSTNmHWENPA
    Vm-BFU012.0001.6180.1900.3840.5700.3350.3090.1471.446***0
    Vm-BFU026.0001.4910.0860.3310.7040.5900.3100.01220.486***1
    Vm-BFU0310.0002.4000.3650.5861.2230.2180.5390.0554.286***9
    Vm-BFU0411.0003.2620.4760.6971.6200.1780.6700.0643.629***3
    Vm-BFU054.0002.1870.2860.5460.9230.3060.4660.01813.956***1
    Vm-BFU065.0002.5950.4170.6181.0750.1790.5470.01813.645***1
    Vm-BFU076.0003.1350.4190.6841.3570.2320.6380.0356.850***2
    Vm-BFU088.0002.1910.5830.5461.1600.0540.5100.0347.124***2
    Vm-BFU094.0002.2370.3900.5560.9080.1620.4530.0723.214***6
    Vm-BFU109.0002.0780.2000.5211.1490.4500.4910.0633.695***12
    Vm-BFU1113.0006.2890.2350.8452.1010.5590.8240.0574.163***3
    Vm-BFU122.0001.8730.3200.4690.6590.1860.3580.0504.742**0
    Vm-BFU1314.0003.1570.4100.6871.6170.2520.6570.0932.439***10
    Vm-BFU1410.0001.8860.2480.4721.1440.3040.4570.0972.338***15
    Vm-BFU1511.0003.1550.3520.6861.5440.3330.6480.0623.808***10
    Vm-BFU169.0005.2530.5480.8141.7870.1930.7820.0564.256***2
    Vm-BFU1714.0003.9130.5380.7481.8520.1580.7250.0317.705***8
    总数 Total138.00048.72885
    平均值 Mean8.1172.8660.3570.5991.2580.2760.5520.0564.2515
    注:Na. 观测到的等位基因数;Ne. 有效等位基因数;Ho. 观测到的杂合度;He. 预期杂合度;I. 香农信息指数;Nm. 基因流;FNull. 无效等位基因频率;PIC. 多态信息含量;FST. 遗传分化指数;HWE. 哈迪−温伯格平衡;*. P < 0.05;**. P < 0.01; ***. P < 0.001;NPA. 私有等位基因的数量。下同。Notes: Na, observed number of alleles; Ne, effective number of alleles; Ho, observed heterozygosity; He, expected heterozygosity; I, Shannon’s information index; Nm, gene flow; FNull, null allele frequency; PIC, polymorphism information content; FST, genetic differentiation index; HWE, Hardy-Weinberg equilibrium; * means P < 0.05; ** means P < 0.01; *** means P < 0.001; NPA, number of private alleles. The same below.
    下载: 导出CSV

    表  5   具有一至多个私有等位基因的千年桐种质

    Table  5   V. montana germplasms with one or more private alleles

    种质
    Germplasm
    群体
    Population
    NPA私有等位基因位点
    Locus of private allele
    种质
    Germplasm
    群体
    Population
    NPA私有等位基因位点
    Locus of private allele
    1-3 1 1 Vm-BFU11 3-3 3 2 Vm-BFU03, Vm-BFU17
    1-4 1 2 Vm-BFU03, Vm-BFU07 3-4 3 3 Vm-BFU03, Vm-BFU14, Vm-BFU16
    1-7 1 2 Vm-BFU03, Vm-BFU07 3-5 3 1 Vm-BFU14
    1-15 1 1 Vm-BFU06 3-6 3 1 Vm-BFU13
    1-18 1 1 Vm-BFU11 3-9 3 3 Vm-BFU03, Vm-BFU11, Vm-BFU13
    1-23 1 1 Vm-BFU13 3-10 3 1 Vm-BFU15
    1-27 1 2 Vm-BFU10, Vm-BFU17 3-12 3 1 Vm-BFU14
    2-2 2 1 Vm-BFU10 3-13 3 1 Vm-BFU15
    2-3 2 1 Vm-BFU09 3-14 3 2 Vm-BFU14, Vm-BFU17
    2-4 2 1 Vm-BFU10 3-15 3 1 Vm-BFU13
    2-5 2 3 Vm-BFU09, Vm-BFU10, Vm-BFU13 3-16 3 1 Vm-BFU14
    2-6 2 2 Vm-BFU03, Vm-BFU13 3-17 3 1 Vm-BFU14
    2-11 2 1 Vm-BFU10 3-22 3 1 Vm-BFU03
    2-13 2 1 Vm-BFU15 3-23 3 2 Vm-BFU04, Vm-BFU14
    2-14 2 2 Vm-BFU10, Vm-BFU15 3-24 3 1 Vm-BFU15
    2-15 2 1 Vm-BFU09 3-25 3 3 Vm-BFU04, Vm-BFU14, Vm-BFU15
    2-16 2 1 Vm-BFU09 3-26 3 1 Vm-BFU14
    2-17 2 1 Vm-BFU09 3-27 3 2 Vm-BFU15, Vm-BFU17
    2-19 2 1 Vm-BFU15 3-28 3 2 Vm-BFU08, Vm-BFU17
    2-20 2 1 Vm-BFU10 3-29 3 1 Vm-BFU14
    2-23 2 1 Vm-BFU10 3-30 3 1 Vm-BFU14
    2-24 2 1 Vm-BFU09 3-31 3 1 Vm-BFU14
    2-26 2 1 Vm-BFU02 3-32 3 2 Vm-BFU03, Vm-BFU05
    2-27 2 2 Vm-BFU10, Vm-BFU15 3-33 3 1 Vm-BFU16
    2-28 2 1 Vm-BFU13 3-35 3 1 Vm-BFU14
    2-29 2 1 Vm-BFU13 10-7 3 1 Vm-BFU17
    2-30 2 2 Vm-BFU10, Vm-BFU13 10-13 3 2 Vm-BFU03, Vm-BFU15
    2-31 2 1 Vm-BFU13 10-16 3 1 Vm-BFU14
    2-33 2 1 Vm-BFU10 10-18 3 1 Vm-BFU10
    3-1 3 1 Vm-BFU17 Guizhou-2 3 2 Vm-BFU08, Vm-BFU17
    3-2 3 1 Vm-BFU14 Guizhou-27 3 1 Vm-BFU04
    总计 Total 85
    下载: 导出CSV

    表  6   千年桐群体间与群体内的AMOVA分析

    Table  6   AMOVA analysis among and within V. montana populations

    变异来源
    Source of variation
    自由度
    Degree of freedom
    方差和
    Sum of squares
    变异组分
    Variance component
    占总变异的比例
    Proportion in total variation/%
    FST
    群体间 Among populations 2 110.841 1.209 8 0.080 (P < 0.001)
    群体内 Within population 102 1 423.025 13.951 92
    总计 Total 104 1 533.867 15.160 100
    下载: 导出CSV
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出版历程
  • 收稿日期:  2021-02-18
  • 修回日期:  2021-03-25
  • 网络出版日期:  2021-10-15
  • 发布日期:  2021-11-29

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