Identification of Natural Hybrids by SSR Markers in Mussaenda

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Journal of Integrative Plant Biology
Oct 2015


Detection of natural hybrids is of great significance for plant taxonomy, reproductive biology, and population genetic studies. Compared with methods depending on morphological characters, molecular markers provide reliable and much more accurate results. This protocol describes approaches employing microsatellite (SSR) markers to identify inter-specific hybrids in Mussaenda (Rubiaceae).

Keywords: Gene flow (基因流), Natural hybridization (自然杂交), SSR (SSR)

Materials and Reagents

  1. Consumables
    1. Microfuge tubes (Corning, Axygen®, catalog number: MCT-150-C )
    2. Pipette tips (Corning, Axygen®, catalog number: T-1000-C , T-200-C , T-300 )

  2. Plant material
    1. Fresh leaves of the putative hybrid individuals, as well as the parental species, were collected and dried in silica gel. It usually takes 3-5 days for complete drying of the leaves.

  3. Chemicals
    1. Taq PCR mastermix (Tiangen, catalog number: KT201 )
    2. Liquid nitrogen
    3. Absolute ethanol (Sinopharm Chemical Reagent, catalog number: 10009228 ) (ice cold)
    4. 70% ethanol (ice cold)
    5. 7.5 M ammonium acetate (Sigma-Aldrich, catalog number: A1542-250G )
    6. Chloroform: isoamyl alcohol (24:1) (Sinopharm Chemical Reagent, catalog number: chloroform, 10006818 ; isoamyl alcohol, 10003218 )
    7. ddH2O (sterile)
    8. 5x Loading buffer with GelRed (Shanghai Generay Biotech, catalog number: GR0205-500 )
    9. Cetyltrimethylammonium bromide/Hexadecyl trimethyl-ammonium bromide (CTAB) (Thermo Fisher Scientific, catalog number: ICN19400480 )
    10. Ethylenediaminetetraacetic acid (0.5M solution/pH 8.0) (EDTA) (Thermo Fisher Scientific, catalog number: BP2482-500 )
    11. Polyvinyl pyrrolidone, MW 40,000 (PVP 40)
    12. NaCl (Sigma-Aldrich, catalog number: S5886-500G )
    13. HCl (Sigma-Aldrich, catalog number: 258148 )
    14. Tris base (Thermo Fisher Scientific, catalog number: BP152-1 )
    15. Boric acid (Sigma-Aldrich, catalog number: B0394-100G )
    16. Agarose (Biowest, catalog number: 111860 )
    17. Hi-DiTM formamide (Thermo Fisher Scientific, Applied BiosystemsTM, catalog number: 4311320 )
    18. GeneScan 500Liz (Thermo Fisher Scientific, Applied BiosystemsTM, catalog number: 4322682 )
    19. CTAB buffer (see Recipes)
    20. 1 M Tris (pH 8.0) (see Recipes)
    21. TE buffer (see Recipes)
    22. 5x TBE (Tris-Borate-EDTA) buffer (Stock) (see Recipes)
    23. 1% agarose gel (see Recipes)


  1. Mortar and pestle
  2. Microcentrifuge (Eppendorf, model: 5427R )
  3. Water bath (IKA, model: HB10 )
  4. PCR thermal cycler (EASTWIN, model: ETC-811 )
  5. Agarose gel electrophoresis system [include PowerPacTM Basic Power Supply for electrophoresis (Bio-Rad Laboratories, catalog number: 1645050 )]
  6. Genetic Analyzer (Invitrogen, model: ABI PRISM 3100 )


  1. GeneMarker version 2.4.0 (
  2. FSTAT version 2.9.3 (
  3. GenAlEx version 6.2 (
  4. STRUCTURE version 2.2 (
  5. NewHybrids version 1.1 beta (


  1. DNA extraction and polymerase chain reaction
    Total DNA was extracted using a modified cetyltrimethylammonium bromide (CTAB) method (Doyle, 1991). Ten pairs of SSR markers were developed by Duan and Zhang (2014) (AC30, CT12, CT59, CT113, CT17, CT60, CT48, CT135, CT142, CT91) from M. pubescens were selected for testing the gene flow between M. pubescens var. alba and M. kwangtungensis. The forward of each primer pair was labeled with fluorochrome (FAM, HEX, ROX, TAMRA). The information of the primers is shown in Table 1.

    Table 1. Characteristics of the 10 pairs of SSR primers employed in this study. Presented for each locus are the forward (F) and reverse (R) primer sequences, repeat motif, size of the original fragment (bp), annealing temperature (Ta), and GenBank accession number. Td, touchdown.

    DNA amplifications were carried out in 10 μl reaction mixtures:
    5 μl Taq PCR mastermix
    0.2 μM of each primer
    3.6 μl ddH2O
    20-40 ng of genomic DNA
    The amplification conditions were as follows:
    1. 94 °C, 4 min (initial denaturation)   
    2. 94 °C, 30 sec (denaturation)
    3. 60 °C, 30 sec (annealing)
    4. 72 °C, 30 sec (extension)
      7 cycles from step (b) to (d)
    5. 94 °C, 30 sec (denaturation)
    6. 53 °C, 30 sec (annealing)
    7. 72 °C, 30 sec (extension)
      28 cycles from step (e) to (g)
    8. 72 °C, 10 min (final extension)
    9. Keep sample at 4 °C until further processing
    The PCR products were determined on an ABI PRISM 3100 Genetic Analyzer in the Sangon Biotech (Shanghai) Co., Ltd., and 1 μl products (10-fold diluted) were mixed with 15 μl Hi-DiTM Formamide probe, which included 2% GeneScan 500Liz. The amplified SSR fragment data were collected using GeneMarker version 2.4.0 software.
  2. Data analysis
    The software FSTAT 2.9.3 (Goudet, 2001) and GenAlEx 6.2 (Peakall and Smouse, 2006) were used for calculating the alleles and private alleles, respectively. We used the program STRUCTURE 2.2 (Pritchard et al., 2000; Falush et al., 2003 and 2007) for population genetic structure analysis and hybrid detection. This method implements a Bayesian model-based clustering approach. We supposed all the loci were independent, following the Hardy–Weinberg equilibrium and linkage equilibrium. The program was run 10 times for each K-value, ranging 1-5, with 100,000 replicates for burn-in and 40,000 iterations. Out of the five runs for k = 2, the run with the highest likelihood value was selected to assign the posterior membership coefficients (Evanno et al., 2005).
    The entire data set is arranged as a matrix in a single file, in which the data for individuals are in rows, and the loci are in columns. For a diploid organism, data for each individual can be stored either as 2 consecutive rows, where each locus is in one column, or in one row, where each locus is in two consecutive columns. Missing data should be indicated by a number that doesn’t occur elsewhere in the data.
    The software NewHybrids version 1.1 beta (Anderson and Thompson, 2002) was used to identify hybrids in the individuals sampled. The program calculates the posterior probability that fall into each of the six genotype categories, Parent 1, Parent 2, F1 hybrids, F2 hybrids, back-cross generation to Parent 1, and back-cross generation to Parent 2.
    For FSTAT, GenAlex and NewHybrids, we used the default value in each software. For STRUCTURE, we chose admixture model, and set the parameters as we mentioned in the original article (Luo et al., 2015), other parameters we used the default values.

Representative data

A representative example of data can be found in our published work (Luo et al., 2015). Figure 1 shows genotype class analysis of all sampled individuals based on the programs NewHybrids and STRUCTURE using SSR data.

Figure 1. Genotype class analysis of all sampled individuals based on the programs NewHybrids (A) and STRUCTURE (B) using SSR data. A. NewHybrids calculated posterior probability that falls into each of six genotype categories: P1 (Parent 1), P2 (Parent 2), F1 (F1 hybrids), F2 (F2 hybrids), BC 1 (backcross generation to Parent 1), BC 2 (backcross generation to Parent 2). B. Structure of 81 accessions based on 13 SSR loci (K = 2). Each bar represents one individual, P1 = M. kwangtungensis, P2 = M. pubescens var. alba.


  1. CTAB buffer, 100 ml
    2.0 g CTAB
    10.0 ml 1 M Tris (pH 8.0)
    4.0 ml 0.5 M EDTA (pH 8.0)
    28.0 ml 5 M NaCl
    40.0 ml H2O
    1 g PVP 40
    Adjust all to pH 8.0 and make up to 100 ml with ddH2O.
  2. 1 M Tris pH 8.0, 1 L
    121.1 g Tris
    700 ml ddH2O
    Dissolve Tris in ddH2O and bring to 900 ml.
    Adjust pH to 8.0 by adding 40-50 ml of concentrated HCl.
  3. TE buffer for 100 ml
    10 mM 1 ml of 1 M Tris (pH 8.0)
    1 mM 0.2 ml of 0.5 M EDTA
  4. 5x TBE (Tris-Borate-EDTA) buffer (Stock)
    54 g Tris base
    27.5 g boric acid
    20 ml 0.5 M EDTA (pH 8.0)
    Adjust pH to 8.3 by HCl.
    This stock solution can be diluted to 1x prior to use in electrophoresis.
  5. 1% agarose gel
    Dissolve 1 g agarose in 100 ml 1x TBE


This work was supported by the National Natural Science Foundation of China (31000109, 31370269, 31170184, U1202261) and the International Foundation for Science (AD/22476). This protocol was modified from our previous work, Luo, Z. L., Duan, T. T., Yuan, S., Chen, S., Bai, X. F., and Zhang, D. X. (2015) Reproductive isolation between sympatric sister species, Mussaenda kwangtungensis and M. pubescens var. alba. J Integr Plant Biol 57: 859-870.


  1. Anderson, E. C. and Thompson, E. A. (2002). A model-based method for identifying species hybrids using multilocus genetic data. Genetics 160(3): 1217-1229.
  2. Doyle, J. (1991). DNA protocols for plants-CTAB total DNA isolation. In: Hewitt, G. M. and Johnston, A. (eds). Molecular techniques in taxonomy. Springer, 283-293.
  3. Duan, T. and Zhang, D. (2014). Fourteen additional microsatellite markers for Mussaenda pubescens and cross-species amplification. J Genet 93(2): e44-47.
  4. Evanno, G., Regnaut, S. and Goudet, J. (2005). Detecting the number of clusters of individuals using the software STRUCTURE: A simulation study. Mol Ecol 14: 2611-2620.
  5. Falush, D., Stephens, M. and Pritchard, J. K. (2003). Inference of population structure using multilocus genotype data: linked loci and correlated allele frequencies. Genetics 164(4): 1567-1587.
  6. Falush, D., Stephens, M., and Pritchard, J. K. (2007). Inference of population structure using multilocus genotype data: Dominant markers and null allele. Mol Ecol Notes 7: 574-578.
  7. Goudet, J. (2001). FSTAT, a program to estimate and test gene diversities and fixation indices (version 2.9.3) (updated from Goudet, J. (1995). FSTAT (version 1.2): a computer program to calculate F-statistics. J. Hered. 86: 485-486). Available at:
  8. Luo, Z. L., Duan, T. T., Yuan, S., Chen, S., Bai, X. F., and Zhang, D. X. (2015) Reproductive isolation between sympatric sister species, Mussaenda kwangtungensis and M. pubescens var. alba. J Integr Plant Biol 57: 859-870.
  9. Peakall, R. and Smouse, P. E. (2012). GenAlEx 6.5: genetic analysis in Excel. Population genetic software for teaching and research--an update. Bioinformatics 28(19): 2537-2539.
  10. Pritchard, J., Stephens, M., and Donnelly, P. (2000). Inference of population structure using multilocus genotype data. Genetics 155: 945-959
  11. Shaw, J., Lickey, E. B., Schilling, E. E. and Small, R. L. (2007). Comparison of whole chloroplast genome sequences to choose noncoding regions for phylogenetic studies in angiosperms: the tortoise and the hare III. Am J Bot 94(3): 275-288.


自然杂交的检测对植物分类学,生殖生物学和群体遗传学研究具有重要意义。 与依赖于形态特征的方法相比,分子标记提供可靠和更准确的结果。 该协议描述了采用微卫星(SSR)标记来鉴定Mussaenda(茜草科)中的特异性杂交体的方法。

关键字:基因流, 自然杂交, SSR


  1. 消耗品
    1. Microfuge管(Corning,Axygen ,目录号:MCT-150-C)
    2. 移液管吸头(Corning,Axygen ,目录号:T-1000-C,T-200-C,T-300)
  2. 植物材料
    1. 收集推定的杂种个体的新鲜叶子以及亲本物种,并在硅胶中干燥。通常需要3-5天完全干燥叶子。

  3. 化学品
    1. Taq PCR mastermix(Tiangen,目录号:KT201)
    2. 液氮
    3. 无水乙醇(国药集团化学试剂,目录号:10009228)(冰冷)
    4. 70%乙醇(冰冷)
    5. 7.5M乙酸铵(Sigma-Aldrich,目录号:A1542-250G)
    6. 氯仿:异戊醇(24:1)(Sinopharm Chemical Reagent,目录号:氯仿,10006818;异戊醇,10003218)
    7. ddH 2 O(无菌)
    8. 5x装载缓冲液,使用GelRed(Shanghai Generay Biotech,目录号:GR0205-500)
    9. 十六烷基三甲基溴化铵/十六烷基三甲基溴化铵(CTAB)(Thermo Fisher Scientific,目录号:ICN19400480)
    10. 乙二胺四乙酸(0.5M溶液/pH 8.0)(EDTA)(Thermo Fisher Scientific,目录号:BP2482-500)
    11. 聚乙烯吡咯烷酮,MW 40,000(PVP 40)
    12. NaCl(Sigma-Aldrich,目录号:S5886-500G)
    13. HCl(Sigma-Aldrich,目录号:258148)
    14. Tris碱(Thermo Fisher Scientific,目录号:BP152-1)
    15. 硼酸(Sigma-Aldrich,目录号:B0394-100G)
    16. 琼脂糖(Biowest,目录号:111860)
    17. Hi-Di TM甲酰胺(Thermo Fisher Scientific,Applied Biosystems TM ,目录号:4311320)
    18. GeneScan 500Liz(Thermo Fisher Scientific,Applied Biosystems TM ,目录号:4322682)
    19. CTAB缓冲区(请参阅配方)
    20. 1 M Tris(pH 8.0)(参见配方)
    21. TE缓冲区(参见配方)
    22. 5x TBE(Tris-硼酸盐-EDTA)缓冲液(储液)(见配方)
    23. 1%琼脂糖凝胶(见配方)


  1. 砂浆和杵
  2. 微量离心机(Eppendorf,型号:5427R)
  3. 水浴(IKA,型号:HB10)
  4. PCR热循环仪(EASTWIN,型号:ETC-811)
  5. 琼脂糖凝胶电泳系统[包括用于电泳的PowerPac TM基本电源(Bio-Rad Laboratories,目录号:1645050)]
  6. 遗传分析仪(Invitrogen,型号:ABI PRISM 3100)


  1. GeneMarker版本2.4.0(。 html
  2. FSTAT版本2.9.3( http://www2.unil。 ch/popgen/softwares/fstat.htm
  3. GenAlEx 6.2版( http://biology-assets。
  4. STRUCTURE版本2.2(
  5. NewHybrids版本1.1测试版( http: //


  1. DNA提取和聚合酶链反应 使用改进的十六烷基三甲基溴化铵(CTAB)方法(Doyle,1991)提取总DNA。由Duan和Zhang(2014)(AC30,CT12,CT59,CT113,CT17,CT60,CT48,CT135,CT142,CT91)从M开发的十对SSR标记。 pubescens 用于测试M之间的基因流。 pubescens var。 和 M。 kwangtungensis 。每个引物对的正向用荧光染料(FAM,HEX,ROX,TAMRA)标记。引物的信息如表1所示
    表1.本研究中使用的10对SSR引物的特征针对每个基因座呈现的是正向(F)和反向(R)引物序列,重复基序,原始片段的大小bp),退火温度(℃)和GenBank登录号。 Td,触地。

    5μlTaq PCR主混合物
    3.6μlddH 2 O 2/d 20-40 ng基因组DNA
    1. 94℃,4分钟(初始变性)   
    2. 94℃,30秒(变性)
    3. 60℃,30秒(退火)
    4. 72℃,30秒(延伸)
    5. 94℃,30秒(变性)
    6. 53℃,30秒(退火)
    7. 72℃,30秒(延伸)
    8. 72℃,10分钟(最终延伸)
    9. 保持样品在4℃直到进一步处理
    PCR产物在Sangon Biotech(上海)有限公司的ABI PRISM 3100遗传分析仪上测定,1μl产物(10倍稀释)与15μlHi-Di TM >甲酰胺探针,其包括2%GeneScan 500Liz。使用GeneMarker版本2.4.0软件收集扩增的SSR片段数据。
  2. 数据分析
    软件FSTAT 2.9.3(Goudet,2001)和GenAlEx 6.2(Peakall和Smouse,2006)分别用于计算等位基因和私有等位基因。我们使用了程序STRUCTURE 2.2(Pritchard等人 ,2000; Falush 等人 )用于群体遗传结构分析和杂交检测。该方法实现了基于贝叶斯模型的聚类方法。我们认为所有的轨迹是独立的,遵循Hardy-Weinberg平衡和连接平衡。该程序对于每个K值运行10次,范围为1-5,对于老化和40,000次迭代具有100,000次重复。在k = 2的五个运行中,选择具有最高似然值的运行以分配后验成员系数(Evanno等人,2005)。
    使用软件NewHybrids版本1.1β(Anderson和Thompson,2002)来鉴定所采样的个体中的杂种。该程序计算落入六个基因型类别(亲本1,亲本2,F1杂种,F2杂种,向亲本1的回交世代和亲本2的回交世代)中的每一个的后验概率。 对于FSTAT,GenAlex和NewHybrids,我们在每个软件中使用默认值。对于STRUCTURE,我们选择混合模型,并设置我们在原始文章(Luo等人。,2015)中提及的参数,其他参数我们使用默认值。



图1.基于使用SSR数据的程序NewHybrids(A)和STRUCTURE(B)的所有样本个体的基因型类分析 A.新的Hybrids计算落入六个基因型类别中的每一个的后验概率:P1 (亲本1),P2(亲本2),F1(F1杂交体),F2(F2杂交体),BC1(与亲本1的回交代),BC2 B.基于13个SSR基因座(K = 2)的81个种质的结构。每个条表示一个个体,P1 = M。 kwangtungensis ,P2 = em。 pubescens var。 。


  1. CTAB缓冲液,100ml
    2.0 g CTAB
    10.0ml 1M Tris(pH8.0) 4.0ml 0.5M EDTA(pH8.0)
    28.0ml 5M NaCl
    40.0ml H 2 O v/v 1 g PVP 40
    将所有pH调节至8.0并用ddH 2 O补足至100ml
  2. 1M Tris pH 8.0,1L
    700ml ddH 2 O
    将Tris溶解在ddH 2 O中并使其达到900ml 通过加入40-50ml浓HCl将pH调节至8.0
  3. TE缓冲液100ml,
    10mM 1ml 1M Tris(pH8.0) 1mM 0.2ml 0.5M EDTA
  4. 5x TBE(Tris-硼酸盐-EDTA)缓冲液(储液)
    27.5克硼酸 20ml 0.5M EDTA(pH8.0) 用HCl调节pH至8.3 在使用电泳之前,该储备溶液可稀释至1x
  5. 1%琼脂糖凝胶 将1g琼脂糖溶解在100ml 1x TBE中


这项工作得到国家自然科学基金(31000109,31370269,31170184,U1202261)和国际科学基金会(AD/22476)的支持。该协议是从我们以前的工作中修改的,Luo,ZL,Duan,TT,Yuan,S.,Chen,S.,Bai,XF和Zhang,DX(2015)Reproductive isolation between sympies sister species,/em> kwangtungensis 和 M。 pubescens var。 。 J Integr Plant Biol 57:859-870。


  1. Anderson,EC和Thompson,EA(2002)。  A基于模型的方法用于使用多基因遗传数据鉴定物种杂种。遗传学160(3):1217-1229。
  2. Doyle,J。(1991)。植物的DNA方案 - CTAB总DNA分离。 In:Hewitt,GM and Johnston,A.(eds)。  分子技术。 Springer ,283-293。
  3. Duan,T。和Zhang,D。(2014)。  用于Mussaenda pubescens的十四种额外的微卫星标记和跨物种扩增。 93 Genet 93(2):e44-47。
  4. Evanno,G.,Regnaut,S.和Goudet,J。(2005)。  使用软件检测个人群集的数量结构:模拟研究 Mol Ecol 14:2611-2620。
  5. Falush,D.,Stephens,M.和Pritchard,JK(2003)。  使用多基因基因型数据的群体结构推断:连锁基因座和相关等位基因频率。遗传学164(4):1567-1587。
  6. Falush,D.,Stephens,M。和Pritchard,JK(2007)。  使用多基因基因型数据推导群体结构:显性标记和无效等位基因。 7:574-578。
  7. Goudet,J。(2001)。 FSTAT,用于估计和测试基因多样性和固定指数的程序(2.9.3版)(从Goudet,J。(1995)。FSTAT(1.2版)更新:计算F-统计的计算机程序J.Hered.86 :485-486)。可在以下网址获取: /izea/softwares/fstat.html。
  8. Lua,ZL,Duan,TT,Yuan,S.,Chen,S.,Bai,XF,and Zhang,DX(2015)  同生姊妹物种,Mussaenda kwangtungensis和 M之间的生殖隔离。 pubescens var。 J Integr Plant Biol 57:859-870。
  9. Peakall,R。和Smouse,PE(2012)。  GenAlEx 6.5:Excel中的遗传分析。用于教学和研究的群体遗传学软件 - 更新。生物信息学28(19):2537-2539。
  10. Pritchard,J.,Stephens,M.,and Donnelly,P.(2000)。  使用多位点基因型数据推断群体结构。 155:945-959
  11. Shaw,J.,Lickey,EB,Schilling,EE and Small,RL(2007)。  比较整个叶绿体基因组序列以选择非编码区域用于被子植物中的系统发育研究:龟和野兔III.The Am J Bot 94(3): 275-288。
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引用:Luo, Z., Duan, T., Yuan, S., Chen, S., Bai, X. and Zhang, D. (2016). Identification of Natural Hybrids by SSR Markers in Mussaenda. Bio-protocol 6(13): e1853. DOI: 10.21769/BioProtoc.1853.