基于EST-SSR标记的栀子品种亲缘关系分析及指纹图谱构建

邓绍勇，祝必琴，李康琴，陈宜均，朱培林，王贤荣，李 婷，唐 山

基于EST-SSR标记的栀子品种亲缘关系分析及指纹图谱构建

邓绍勇1, 2，祝必琴3，李康琴1, 2，陈宜均1, 2，朱培林1, 2，王贤荣4，李 婷1, 2，唐 山1

1.江西省林业科学院，江西 南昌 330032 2.国家林业草原栀子工程技术研究中心，江西 南昌 330032 3.江西省森林资源保护中心，江西 南昌 330038 4.南京林业大学生物与环境学院，江苏 南京 210037

使通过分子标记手段鉴定栀子品种成为现实，满足苗期鉴别栀子品种资源的需要。采用14对多态性较好的转录组微卫星（EST-SSR）引物对栀子及10个品种进行扩增检测，分析其遗传多样性和遗传距离等参数，并进行系统聚类。14对EST-SSR引物共检测到62个等位基因，平均每个位点扩增出4.4条，栀子品种Nei多样性指数（）和Shannon指数（）分别为0.653 3和1.226 8，表现出较高的遗传多样性水平。同一品种的不同样品能较好地聚集在一起，但品种之间并未完全按形态学性状聚类分支。构建的指纹图谱通过引物及引物组合能较好地将栀子品种进行区分。利用14对EST-SSR引物成功地构建了10个栀子品种的指纹图谱，研究结果可为栀子品种鉴定、亲缘关系及品种起源提供科学依据。

栀子；EST-SSR；指纹图谱；遗传多样性；亲缘关系

栀子Ellis 为茜草科栀子属常绿灌木，其花洁白如雪、香如凝脂，是我国著名的八大香花之一[1]，栀子果实还是大宗的中药材原料，入药具有泻火除烦、清热利湿、凉血散淤的功效，现代药理研究发现栀子具有保肝、抗炎、镇痛及改善心脑血管病等方面的作用[2]。栀子在长期的栽培利用过程中，使其习性、花、叶的形状及大小、果实的形状及大小等均发生了丰富的变异，在色素用、药用和观赏等不同利用方向都形成了稳定的变异类型[3-4]，国内外一些研究者命名了不同的变种或变型[5-6]，也有前人对栀子品种进行了系统的分类整理[7]，并从数量分类视角对栀子栽培品种与近缘种亲缘关系进行了探讨[8]。为规避环境因子和人为主观意识等的干扰，有研究者采用分子标记法对栀子不同栽培类型进行鉴别以及研究它们之间的亲缘关系，杨锐培[9]采用RAPD标记分析了江西樟树栀子基地形态上区别明显的14个桅子样品，发现分子聚类结果和表型聚类结果类似；韩建萍等[10]采用AFLP标记分析了江西5个不同产地栀子的亲缘关系，发现各地方样品间遗传变异明显，且样品间遗传距离和地理距离相关性明显。葛菲等[11-12]采用RAPD标记分析了栀子与雀舌栀子、重瓣栀子及水栀子间的亲缘关系，结果显示重瓣栀子与栀子遗传距离最近，雀舌栀子与栀子遗传距离最远。鲁耀邦等[13]采用RAPD标记分析了桅子与水桅子，野生桅子与栽培桅子间的DNA多态性。

SSR分子标记是以扩增片段的长度变异作为多态性的共显性分子标记。由于微卫星序列重复的长度及数目变化极其丰富，而使这种多态性检测方法极为有效，目前已广泛用于植物各水平遗传多样性、分子系统学、品种和纯度鉴定、基因定位、遗传图谱构建、比较基因组学等各方面研究中。在对植物种下等级亲缘关系、种质鉴定研究中，相对其它分子标记表现出明显的优越性[14-16]。本实验对栀子常见主栽品种构建了转录组微卫星分子标记（transcriptome microsatellite markers，EST-SSR）指纹图谱，以遗传距离矩阵为分析对象，按不加权成组配对法（UPGMA）进行聚类分析，建立栀子品种间的亲缘关系树状图。系统探讨栀子栽培品种遗传距离和亲缘关系，以期为栀子品种鉴定、亲缘关系及品种起源提供科学依据。

1 材料与方法

1.1 材料

实验材料为10个栀子品种[7]和1个栀子栽培原种共39份，每个品种1～5份样本。所有实验材料由江西省林业科学院邓绍勇副研究员鉴定为栀子Ellis及其不同的品种类型，采集新鲜幼嫩叶片放入自封袋内，随即加入变色硅胶并摇匀（变色硅胶与样品质量比约为10∶1），封好袋后于常温干燥处保存。根据硅胶变色情况及时加换硅胶。样品来源等基本情况详见表1。

表1 栀子品种取样材料

Table 1 Information on ten cultivars of G.jasminoides

编号品种名称品种来源地编号品种名称品种来源地 1宽棱水栀1江西省樟树市吴城药材基地种植21金福水栀1湖北省宜昌市金福村栽培 2荷花栀子1广东省广州市园林栽培22金福水栀2湖北省宜昌市金福村栽培 3荷花栀子2广东省广州市园林栽培23金福水栀3湖北省宜昌市金福村栽培 4荷花栀子3广东省广州市园林栽培24太湖山栀1安徽省潜山太湖 5荷花栀子4广东省广州市园林栽培25太湖山栀2安徽省潜山太湖 6荷花栀子5广东省广州市园林栽培26山栀子1江西省永丰县瑶里 7大花栀子1重庆市27山栀子2江西省永丰县瑶里 8大花栀子2重庆市28山栀子3江西省永丰县瑶里 9大花栀子3重庆市29山栀子4江西省永丰县瑶里 10小白蟾1广东省东莞市园林栽培30山栀子5江西省永丰县瑶里 11小白蟾2广东省东莞市园林栽培31球果栀子1江西省樟树市隍城栀子GAP药材基地种植 12小白蟾3广东省东莞市园林栽培32球果栀子2江西省樟树市隍城栀子GAP药材基地种植 13雀舌栀子1湖北省武汉市园林栽培33白蟾1江西省南昌市园林栽培 14雀舌栀子1江西省南昌市园林栽培34白蟾2江西省南昌市园林栽培 15雀舌栀子2江西省南昌市园林栽培35白蟾3江西省南昌市园林栽培 16雀舌栀子3江西省南昌市园林栽培36小白蟾A1广东省东莞市园林栽培 17水栀子1江西省樟树市吴城药材基地种植37小白蟾A2广东省东莞市园林栽培 18水栀子2江西省樟树市吴城药材基地种植38小白蟾B1广东省东莞市园林栽培 19水栀子3江西省樟树市吴城药材基地种植39小白蟾B2广东省东莞市园林栽培 20水栀子4江西省樟树市吴城药材基地种植

1.2 方法

采用改进的CTAB裂解-硅珠吸附法从变色硅胶保存的叶片中提取总基因组DNA，从25对栀子EST-SSR多态引物[17]中筛选出14对适用于栀子品种扩增的多态引物（表2），所有的PCR扩增反应程序由Eppendorf 5331梯度PCR扩增仪（Eppendorf，Hamburg，Germany）执行。反应体系为：10 μL中含1.5 μL模板DNA（50 ng/μL），1 μL 10×Buffer（Mg2+free），0.6 μL MgCl2（25 mmol/L），0.8 μL dNTPs（10 mmol/L），0.3 μL F-primer（10 μmol/L），0.3 μL R-primer（10 μmol/L），0.1 μL Taq酶（5 U/μL）（TaKaRa Bio Inc.，Otsu，Shiga公司，日本），5.4 μL ddH2O；反应程序为：94 ℃预变性3 min；94 ℃变性30 s，63 ℃退火30 s（每循环降低1 ℃），72 ℃延伸30 s，共15个循环；94 ℃变性30 s，57 ℃退火30 s，72 ℃延伸30 s，共15个循环；72 ℃延伸10 min；10 ℃保持。PCR扩增产物用8%聚丙烯酰胺凝胶电泳检测，采用银染技术显带。

表2 14对栀子EST-SSR引物

Table 2 Fourteen pairs of EST-SSR primers for amplifying microsatellite loci of G.jasminoides

位点引物序列(5'→3')重复单元片段大小/bp退火温度/℃GenBank 登记号 eGJ004F:TCACCTCAGCAGCCTACTCT(CCAAA)519960KM279432 R:AGAGAGAAGTGGGCACGTTG eGJ006F:CATGCCGCCGTGATTGATTT(AATAG)516860KM279433 R:GCCCCTTCTAGCCAATGTGT eGJ011F:CTCCTGGGCAAGACTTTGGT(TTGCC)522160KM279435 R:GCGGTGGATCGGTGTCTTAA eGJ015F:CCAAACCAACCGCTCAACAG(ACTC)612760KM279436 R:ATGCCGTGTGTTGAGTGAGT eGJ019F:GAAGGGGTGGCTCCAATTCA(GCTG)527760KM279437 R:ACTTGGTCCGCTGGATAACG eGJ026F:GTCACTGTGGCTGCCATTTG(GCT)716460KM279438 R:AGAGCAACGCCACGTATCAA eGJ041F:CTCCCACACACACGGAAAGA(GA)1119760KM279441 R:GCTCAGTGGTGGAGAAGAGG eGJ087F:GCACCAGTTACTTGGTCTCCA(TCC)619160KM279445 R:GAGGGGTTTGCACGTCTGTA eGJ091F:CCCCCACATTCGGCATTTTC(TCA)714260KM279446 R:TCAGTGATTCGTTGGACCCG eGJ098F:GTGGTTCCGGCTAGTCAGTC(GGA)610560KM279447 R:ACCTGCCATCGGATAAAGCC eGJ123F:GAATGCTGTTCCAAAGCTGCA(GA)918360KM279451 R:CTTTGTGGTTCCGTGGCAAG eGJ134F:AAACAGACCAGGCATGAGCA(CA)917560KM279454 R:AAGTGGCACCGTCTGATGAG eGJ144F:TGAGCTGATGCACTCACAGA(AG)922259KM279455 R:CCTGGACCTGGAGAAAGACG eGJ145F:CCCCTTGGTCTAGAGGTGGA(AG)920260KM279456 R:TTAGCCTACGAGGAATGGCG

1.3 数据分析方法

采用多人判读比对方式对胶图数据进行判读，按条带长度大小从大到小用A、B、C、D、E…进行编号，读带结果如AB、BC等，若某一个样本只有一条带，读带结果则为AA、BB等。同时统计各位点条带的片段大小，方便数据数值转换，条带分析完毕之后把所有的结果根据软件格式要求输入相应软件进行实验的数据处理与统计分析。利用POPGEN32软件[18]计算观察等位基因平均数（a）、有效等位基因数目（e）、观察杂合度（o）、期望杂合度（e）、Shannon多样性指数（Nei's genetic identity，）、Nei多样性指数（ei）以及遗传距离（genetic distance，D）。采用NTSYS-pc 2.10e 软件[19]的UPGMA法利用N遗传距离（1978）构建系统聚类图。

2 结果与分析

2.1 栀子品种遗传多样性分析

14对栀子EST-SSR引物均在供试栀子品种样品中得到了较好的多态扩增，所用引物信息详见表2。10个栀子品种和1个栀子栽培原种共39份材料，14对EST-SSR引物共检测到62个等位基因，每个位点实际扩增N2～8个，引物eGJ15扩增等位基因最多为8个，平均为4.43；N为1.52～5.69，N为3.24。39个品种样品的为1.23，N为0.65，观测杂合度（）变化范围为0.15～0.85，平均为0.51；期望杂合度（）变化范围为0.35～0.83，平均为0.66（表3），结果显示栀子品种有着较高的遗传多样性水平。引物eGJ19、eGJ26、eGJ134对栀子39份品种样品的PCR扩增电泳检测结果见图1。

表3 栀子品种14个SSR位点的遗传多样度

Table 3 Genetic diversity of 14 SSR loci within cultivars of G.jasminoides

基因样本数NaNeIObs_HetExp_HetNeiAve_Het eGJ46842.321 30.974 70.617 60.577 70.569 20.269 2 eGJ67453.631 31.430 50.459 50.734 50.724 60.217 9 eGJ116632.535 51.004 70.303 00.614 90.605 60.128 2 eGJ157885.686 01.856 80.256 40.834 80.824 10.128 2 eGJ197873.992 11.548 70.717 90.759 20.749 50.359 0 eGJ267821.517 20.524 30.230 80.345 30.340 90.115 4 eGJ417842.165 10.973 50.410 30.545 10.538 10.205 1 eGJ877832.169 80.911 10.153 80.546 10.539 10.076 9 eGJ917854.127 51.486 90.769 20.767 60.757 70.384 6 eGJ987842.433 61.020 50.282 10.596 70.589 10.141 0 eGJ1236843.184 61.245 00.852 90.696 20.686 00.371 8 eGJ1347854.167 11.502 60.794 90.769 90.760 00.397 4 eGJ1447443.840 11.363 50.756 80.749 70.739 60.359 0 eGJ1457443.612 11.332 20.567 60.733 10.723 20.269 2 平均值754.42863.241 71.226 80.512 30.662 20.653 30.244 5 方差 1.55491.113 20.342 90.242 00.130 80.129 10.114 8

2.2 栀子品种间遗传关系分析

样品间的遗传距离在−0.118～2.859，平均为0.761；相似系数在0.057～1.125，平均为0.497（表4），大花栀子和山栀子间遗传距离最大。以遗传距离矩阵为分析对象，利用NTSYS-pc 2.1软件按UPGMA法进行聚类分析，建立10个栀子品种和1个栀子栽培原种共39份材料间的亲缘关系树状图（图2）。从聚类结果来看，除栽培栀子即山栀子5个样本、金福水栀3个样本及大花栀子未完全聚集在一起，尤其是金福水栀3个样本聚类较为分散，其他8个品种各样本均聚集在一起。其中宽棱水栀显示和其他品种亲缘关系最远，荷花栀子显示和太湖山栀、白蟾亲缘关系较近，球果栀子显示和山栀子亲缘关系较近，小白蟾显示和水栀子亲缘关系较近，大花栀子、雀舌栀子聚类显示则较为独立。

图1 部分SSR引物对栀子39份品种样品的PCR扩增电泳检测

表4 栀子品种间的D和I矩阵

Table 4 D and I matrices among Gardenia varieties

编号123456789101112131415161718192021222324252627282930313233343536373839 1****0.450.470.320.280.270.330.190.190.510.430.530.350.390.350.480.340.500.430.380.280.230.290.330.280.230.350.270.350.220.260.400.270.380.360.370.530.410.56 20.80****0.990.700.830.800.330.440.400.320.300.270.640.830.550.570.380.410.430.380.460.440.410.900.750.620.460.500.760.710.410.540.720.720.690.360.300.220.38 30.750.01****0.720.860.780.240.440.400.350.330.300.620.700.530.540.440.410.430.330.410.520.410.810.720.720.560.610.630.660.460.680.640.750.670.390.330.370.54 41.140.360.32****0.880.850.320.700.620.530.530.550.620.450.380.380.610.570.360.420.290.400.430.730.650.470.360.430.640.540.500.660.640.670.640.350.290.360.37 51.260.190.150.13****1.090.380.580.480.410.390.360.700.570.500.490.440.470.370.380.440.410.440.670.620.490.310.390.540.560.460.570.580.620.610.270.210.340.35 61.300.220.250.16-0.1****0.370.560.470.390.380.350.670.550.480.480.430.460.360.370.420.350.490.670.650.410.240.320.530.540.450.490.590.650.640.260.200.270.28 71.101.111.441.140.971.01****0.390.350.510.430.480.330.320.280.330.310.440.480.290.100.230.470.280.170.060.110.070.290.270.070.250.120.170.200.370.430.330.48 81.650.830.830.350.540.570.95****0.660.500.410.470.410.350.410.360.550.590.490.540.290.330.320.500.530.410.250.300.360.420.400.620.500.390.420.330.360.360.32 91.670.910.910.480.730.761.040.42****0.640.600.600.620.490.470.570.630.740.480.600.390.520.300.610.430.420.340.390.510.410.490.510.580.540.690.570.540.400.48 100.671.141.060.630.900.940.670.700.45****1.111.110.520.420.480.480.680.830.550.710.300.500.380.380.290.280.430.480.550.440.380.400.360.550.500.820.950.770.79 110.851.201.100.640.930.970.850.890.50−0.1****1.130.540.440.390.390.610.700.450.620.310.470.400.390.330.330.490.530.510.400.460.380.410.540.490.720.890.730.75 120.631.301.200.591.011.050.730.760.50−0.1−0.1****0.480.380.390.500.610.730.480.650.260.470.400.360.300.330.490.500.510.400.430.420.380.510.460.750.920.770.79 131.040.440.480.480.360.401.110.880.480.650.610.73****0.880.790.640.510.440.370.440.670.490.210.580.460.420.150.310.450.530.380.340.530.590.720.420.240.220.32 140.950.190.360.800.560.601.141.040.710.870.830.970.13****0.760.670.460.490.520.530.600.480.260.670.540.530.270.460.610.660.530.490.670.670.750.410.350.330.43 151.060.590.640.960.690.731.280.900.750.730.950.950.240.27****0.730.530.490.420.480.560.460.230.430.370.380.120.270.390.480.320.390.440.500.680.450.390.360.47 160.740.570.610.960.710.741.121.030.570.730.940.690.440.400.32****0.410.520.340.380.420.430.270.480.370.450.170.280.360.420.430.440.440.520.640.420.470.310.55 171.090.970.810.490.810.851.180.600.460.380.500.500.680.780.640.89****0.910.740.740.520.430.310.550.510.420.290.560.640.480.580.640.620.630.580.610.500.510.49 180.700.880.880.560.750.780.810.530.300.190.360.310.810.720.710.660.10****0.880.930.430.460.290.510.410.320.270.550.670.530.450.530.520.590.610.630.770.610.66 190.860.860.861.031.001.030.740.720.730.600.800.731.000.650.871.080.300.13****0.870.510.270.190.460.430.450.260.590.510.620.400.450.470.430.400.610.640.590.57 200.950.961.120.860.961.001.240.610.510.350.490.440.830.640.730.980.300.070.14****0.540.420.240.530.490.400.350.620.550.600.500.460.590.490.560.620.650.600.58 211.260.780.881.240.830.862.291.230.941.211.171.350.410.510.570.860.660.840.670.62****0.380.230.470.460.450.190.410.260.370.470.290.550.410.550.520.310.210.27 221.480.820.660.920.881.051.481.110.660.690.760.760.710.740.780.850.850.781.300.860.97****0.400.500.490.600.540.480.640.490.470.550.590.670.730.530.470.570.58 231.250.880.880.850.810.720.761.131.220.960.920.921.581.361.461.301.161.231.671.421.480.92****0.610.530.390.510.340.330.310.540.530.370.500.540.430.400.310.42 241.100.110.210.320.410.401.270.700.500.980.941.020.550.400.850.730.600.670.780.630.750.690.50****0.980.740.570.660.820.600.590.580.840.780.880.490.330.270.45 251.260.290.320.440.480.441.800.630.851.241.101.200.790.611.000.990.680.880.860.710.780.710.630.02****0.720.590.670.630.610.630.600.950.830.870.330.270.250.41 261.470.480.340.750.720.902.860.880.861.271.111.110.860.630.960.800.861.130.800.920.810.520.930.300.34****0.710.670.510.530.730.760.710.590.600.440.370.490.50 271.040.770.591.031.171.432.221.381.080.830.720.721.871.312.111.771.231.301.341.061.680.620.670.570.530.34****0.770.560.500.600.620.640.530.470.450.450.570.59 281.310.690.490.840.941.132.611.200.940.730.630.691.180.781.301.270.580.590.530.470.900.731.090.410.400.390.26****0.600.580.570.500.720.610.540.410.440.610.66 291.060.280.450.440.610.641.231.030.670.590.670.670.790.490.951.020.440.410.670.591.350.441.100.200.450.670.580.52****0.690.490.540.750.760.720.580.510.520.50 301.490.340.410.610.580.611.310.880.900.820.910.910.630.410.740.860.730.630.480.520.990.711.170.500.490.640.690.540.37****0.410.510.660.710.660.490.430.350.45 311.330.890.770.690.770.802.630.930.710.960.770.840.960.641.130.850.540.800.910.690.760.750.610.530.470.320.510.570.710.88****0.890.790.680.710.430.370.570.42 320.920.620.390.420.570.711.390.480.670.920.960.881.080.710.940.810.450.630.800.771.230.600.630.540.520.280.470.690.610.670.11****0.680.650.610.380.380.620.54 331.310.320.450.440.540.532.100.680.541.020.900.980.640.400.830.830.470.650.750.520.600.531.000.170.060.340.440.330.280.410.230.39****0.920.900.470.340.420.49 340.970.320.290.400.480.441.800.940.620.600.610.670.530.400.690.660.460.530.860.710.880.400.690.250.190.540.640.490.280.340.380.430.09****1.010.540.480.400.57 351.030.360.410.440.500.441.590.870.360.690.710.780.320.280.380.450.540.500.920.580.600.320.610.130.140.520.760.620.330.420.350.490.10−0.01****0.490.420.330.51 360.991.010.931.051.301.340.991.110.550.200.330.290.860.890.810.870.500.460.500.490.660.640.840.711.100.820.790.900.550.720.840.960.750.610.71****0.990.770.79 370.631.201.101.231.551.590.851.030.610.050.120.081.421.050.950.750.690.270.450.441.170.760.921.121.301.000.790.820.670.841.000.961.060.730.860.01****0.870.97 380.891.530.991.031.081.311.121.010.910.260.310.261.531.111.011.160.670.490.530.511.550.571.181.321.400.720.560.490.651.040.570.470.870.911.100.260.14****0.95 390.580.970.621.001.051.290.741.150.740.240.290.241.150.850.760.590.720.410.560.541.300.540.870.810.890.690.540.420.690.790.870.620.710.560.670.240.040.05****

对角线以上为，对角线以下为

is above diagonal andis below diagonal

图2 10个栀子品种聚类图

2.3 栀子品种SSR指纹图谱构建

根据多态引物扩增的电泳结果，采用在相同迁移率位置上有带记为“1”，无带记为“0”的读带方法，以14个SSR标记构建栀子10个品种的指纹图谱（表5），由表5可见，引物eGJ15和eGJ19多态性最为丰富，它们组合一起基本可以把10个栀子品种区分开，其他引物通过3个或3个以上组合一起也可以完成对栀子所有品种的区分。

3 讨论

栀子在观赏栽培和果用栽培方面都保留有丰富的品种资源[7]，对栀子品种进行科学规范的分类整理，研究掌握栀子品种遗传多样性和遗传关系，明晰主栽栀子品种起源和系统进化关系，可以为栀子品种鉴定和栀子种质资源创制提供科学依据。本实验采用14对栀子EST-SSR引物对10个栀子品种和1个栀子栽培原种共39份材料进行扩增，共检测到62个等位基因，39个品种样品的为1.23，N为0.65，高于同样栽培历史悠久的观赏海棠[16]和油茶[15]等木本植物，体现出栀子品种具有丰富的遗传多样性。在对栀子品种遗传距离研究方面，葛菲等[11-12]采用RAPD标记分析显示重瓣栀子（白蟾）与栀子遗传距离最近，雀舌栀子与栀子遗传距离最远，本研究结论也显示山栀子与白蟾遗传距离相对山栀子与雀舌栀子遗传距离要更近，且大花栀子和山栀子间遗传距离最大。

杨锐培[9]采用RAPD标记分析了江西樟树吴城乡康成栀子基地形态上区别明显的14个桅子样品，14个样品明显区分成2组，发现分子聚类结果和表型聚类结果类似。以叶、花、果形态学性状为数据基础的数量分类视角对栀子品种进行聚类时，基本按色素用、药用和观赏用形成聚集分支[8]。而采用EST-SSR分子标记的栀子品种聚类并没有得出类似的结果，但除了山栀子5个样本、金福水栀3个样本及大花栀子未完全聚集在一起，尤其是金福水栀3个样本聚类较为分散，其他8个品种各样本均聚集在一起。其中宽棱水栀显示和其他品种亲缘关系最远，荷花栀子显示和太湖山栀、白蟾亲缘关系较近，球果栀子显示和山栀子亲缘关系较近，小白蟾显示和水栀子亲缘关系较近，大花栀子、雀舌栀子聚类显示则较为独立。

植物品种鉴定的可靠性受到多种情况的影响，如通过表型鉴定往往受鉴定者经验、材料完整程度、季节及材料特性等影响，而通过DNA指纹识别来完成品种鉴定可以克服上述一些因素的影响。采用SSR标记构建植物品种指纹具备多态性高、带型清晰、稳定性和重复性好、操作简单、成本低等优点。文雁成等[20]通过比较研究认为SSR标记比SRAP标记更适合以引物组合法构建品种指纹图谱。因此，SSR标记构建的指纹图谱可作为栀子品种间区别的有力参考。本研究根据多态引物扩增的电泳结果，以14个SSR标记构建栀子10个品种的指纹图谱，通过引物指纹的两两组合，或者多个引物指纹的组合，可以达到区分品种的目前，其中引物eGJ15和eGJ19多态性最为丰富，它们组合一起基本可以把10个栀子品种区分开，使采用分子手段鉴定栀子品种成为现实。

表5 10个栀子品种14个SSR标记的指纹图谱

Table 5 Fourteen SSR fingerprints of ten cultivars of G.jasminoides

编号品种中名标记代码 eGJ4eGJ6eGJ11eGJ15eGJ19eGJ26eGJ41eGJ87eGJ91eGJ98eGJ123eGJ134eGJ144eGJ145 1宽棱水栀100010000100101000000000010001010000100110000100011001000011001 2荷花栀子100110100001000001000001100010010000100110000110010101010100110 3荷花栀子200110100001000010000001100010010000100110000110011001010100110 4荷花栀子300110100001001000000001100010010001001000010010011001010100110 5荷花栀子400110100001000010100001100010010000101000010010010100110100110 6荷花栀子500110100001000000100001100010010000101000010010010100110100110 7大花栀子100111010001000001000000000101010010010000010001000000100011001 8大花栀子201100100001000100000000100110000101001000010001010001000010101 9大花栀子300110010010000100000000100110011001001010010011000001011000011 10小白蟾100110001010100100010001010011010001010100010001011010001010001 11小白蟾200110001010110000010001010011011001010100010001011010001100001 12小白蟾300110001010101000001001010011011001010100010001011010001010001 13雀舌栀子100100110010001000010010100010010000100101010001010101000101010 14雀舌栀子100100110010000001000010100010010000100101000101010101000100101 15雀舌栀子200100110010000000010010100010010000100101001001010101000010101 16雀舌栀子300010010010000000001000100010010000100100010101010101000010101 17水栀子100101000110100100010000101010110001011000010101011000010010001 18水栀子200111000110100100000000010110010001011000010101011100010010001 19水栀子300101000110100100000000010110110010011000000101011100010010001 20水栀子400101000110100100000000010010110001011000100101010100010010001 21金福水栀100100000110000100000000100010100000110100001000010100000101010 22金福水栀201000101010000010000100100010010001000110001111000100101001000 23金福水栀311001010001000000100001100001011001010100001100110000101000110 24太湖山栀100110110001000001100010100010110001000110000110010101010100010 25太湖山栀200110100001000000100010100010100001000110000110010101010101100 26山栀子110100101001000010000000100010101011000101000110010101011000110 27山栀子210100101001000010000001000001101001000110000110010101011000110 28山栀子310100101001000010000001000010100001000110000110011100010100001 29山栀子400110101001000001000011000010010001001010000110011100011001010 30山栀子500100100001000100001001000010010011000110000101000100010100100 31球果栀子110100011010000010100000100011101001001000000100011100010100100 32球果栀子210100100111000010000000100011011001001000000101011001010010100 33白蟾100110100010000001100001100010100001001010000110010101010101100 34白蟾200110100010000000100001100010010001000110000110011010010101100 35白蟾300110110010000000100010100010010001000110001110010101010101100 36小白蟾A100110001010000100000001010011110011010100001101101010001010010 37小白蟾A200010001010100100000001010011011011010100001101101010001010001 38小白蟾B100010001010100010000001010011110011001000001101101010001010001 39小白蟾B200010110010100010000001010011110011010100001101101010001010001

利益冲突 所有作者均声明不存在利益冲突

[1] 俞香顺, 周茜.中国栀子审美文化探析 [J].北京林业大学学报: 社会科学版, 2010, 9(1): 6-12.

[2] 李晓雅, 吴敏, 王松子, 等.中药栀子有效成分栀子苷的心血管系统药理作用研究 [J].中国中西医结合杂志, 2022, 42(3): 373-378.

[3] 曹岚, 刘德文, 戴泽霞.江西省栀子种质资源现状调查与分析 [J].时珍国医国药, 2008, 19(2): 288-290.

[4] 周昌华, 张兴翠, 罗健, 等.山栀子品种资源的综合评价 [J].中国中药杂志, 1998, 23(3): 141-141, 188.

[5] 谢宗万.水栀子的品种考证及品质评价刍议 [J].中药材, 1991, 14(7): 45-47.

[6] 郑朝宗 浙江植物志(第六卷) [M].杭州: 浙江科学技术出版社, 1993: 236.

[7] 邓绍勇, 朱培林, 王贤荣.栀子品种分类研究 [J].南方林业科学, 2018, 46(1): 13-18.

[8] 邓绍勇, 杨欢, 朱培林, 等.栀子栽培品种与近缘种的数量分类 [J].江西农业大学学报, 2020, 42(1): 92-100.

[9] 杨锐培.基地栀子优良种质的筛选与高产栽培技术研究 [D].广州: 广州中医药大学, 2014.

[10] 韩建萍, 陈士林, 张文生, 等.栀子道地性的分子生态学 [J].应用生态学报, 2006, 17(12): 2385-2388.

[11] 葛菲.栀子及其近缘类群的随机扩增多态DNA分析 [J].江西中医学院学报, 2007, 19(2): 54-55.

[12] 葛菲, 周至明.栀子及其近缘类群的随机扩增多态DNA分析 [J].时珍国医国药, 2007, 18(8): 1917-1918.

[13] 鲁耀邦, 刘平安, 李彬, 等.栀子DNA指纹图谱的RAPA分析[J].湖南中医药大学学报, 2007, 27: 272-274.

[14] 罗清, 於艳萍, 卢业飞, 等.基于SSR标记的杜鹃品种亲缘关系分析 [J].北方园艺, 2017(19): 111-117.

[15] 周文才, 温强, 杨军, 等.油茶栽培品种SSR指纹图谱构建及聚类分析 [J].分子植物育种, 2017, 15(1): 238-249.

[16] 荣浩, 黄彬, 周琦, 等.61个观赏海棠品种的SSR指纹图谱构建及遗传多样性分析 [J].南京林业大学学报: 自然科学版, 2018, 42(3): 45-50.

[17] Deng S Y, Wang X R, Zhu P L,.Development of polymorphic microsatellite markers in the medicinal plant(Rubiaceae) [J]., 2015, 58: 149-155.

[18] Yeh F C, Yang R C, Boyle T.PopGene Version 131: Microsoft Window-based freeware for population genetic analysis [J]., 1999(3): 11-23.

[19] Rohlf F J.NTSYS-pc: Numerical Taxonomy and Multivariate Analysis System [M].New York: Applied Biostatistics, 1992.

[20] 文雁成, 王汉中, 沈金雄, 等.SRAP和SSR标记构建的甘蓝型油菜品种指纹图谱比较 [J].中国油料作物学报, 2006, 28(3): 233-239.

Genetic relationship analysis and fingerprint construction of Gardenia jasminoides cultivars based on EST-SSR markers

DENG Shao-yong1, 2, ZHU Bi-qin3, LI Kang-qin1, 2, CHEN Yi-jun1, 2, ZHU Pei-lin1, 2, WANG Xian-rong4, LI Ting1, 2, TANG Shan1

1.Forest Academy of Jiangxi Province, Nanchang 330032, China 2.Engineering Research Center for Gardenia of National Forestry and Grassland Administration, Nanchang 330032, China 3.Jiangxi Forest Resources Protection Center, Nanchang 330038, China 4.College of Biology and the Environment, Nanjing Forestry University, Nanjing 210037, China

To identifycultivars by molecular markers, and meet the needs of identifying.cultivars resources at seedling stage.Fourteen pairs of EST-SSR primers with good polymorphism were used to amplify 10.cultivars.The parameters of genetic diversity and genetic distance were analyzed, and the cluster analysis was carried out.A total of 62 alleles were detected by 14 pairs of EST-SSR primers, with an average of 4.4 alleles per locus.The Nei diversity index () and Shannon index () of.cultivars were 0.6533 and 1.2268, showing a high level of genetic diversity.Different samples of the same cultivars could gather together well, but the cultivars did not cluster according to morphological characters.The constructed fingerprints can be used to distinguish.cultivars by primers and primer combinations.Using 14 pairs of EST-SSR primers, the fingerprints of 10 Gardenia cultivars were successfully constructed.The results can provide scientific basis for the identification, genetic relationship and origin of.cultivars.

Ellis; EST-SSR; fingerprint; genetic diversity; genetic relationship

R286.2

A

0253 - 2670(2022)09 - 2795 - 08

10.7501/j.issn.0253-2670.2022.09.023

2021-10-09

国家自然科学基金资助项目（32060356）；国家自然科学基金资助项目（31760220）；中央财政林业科技推广示范项目（JXTG[2022]12号）；江西省重点研发计划项目（20203BBF63024）

邓绍勇（1982－），男，博士，副研究员，主要研究方向为药用植物资源开发与利用。Tel: (0791)83833641 E-mail: jxforestry@163.com

[责任编辑 时圣明]