基于EST-SSR標記的梔子品種親緣關(guān)系分析及指紋圖譜構(gòu)建

鄧紹勇，祝必琴，李康琴，陳宜均，朱培林，王賢榮，李 婷，唐 山

基于EST-SSR標記的梔子品種親緣關(guān)系分析及指紋圖譜構(gòu)建

鄧紹勇1, 2，祝必琴3，李康琴1, 2，陳宜均1, 2，朱培林1, 2，王賢榮4，李 婷1, 2，唐 山1

1.江西省林業(yè)科學(xué)院，江西 南昌 330032 2.國家林業(yè)草原梔子工程技術(shù)研究中心，江西 南昌 330032 3.江西省森林資源保護中心，江西 南昌 330038 4.南京林業(yè)大學(xué)生物與環(huán)境學(xué)院，江蘇 南京 210037

使通過分子標記手段鑒定梔子品種成為現(xiàn)實，滿足苗期鑒別梔子品種資源的需要。采用14對多態(tài)性較好的轉(zhuǎn)錄組微衛(wèi)星（EST-SSR）引物對梔子及10個品種進行擴增檢測，分析其遺傳多樣性和遺傳距離等參數(shù)，并進行系統(tǒng)聚類。14對EST-SSR引物共檢測到62個等位基因，平均每個位點擴增出4.4條，梔子品種Nei多樣性指數(shù)（）和Shannon指數(shù)（）分別為0.653 3和1.226 8，表現(xiàn)出較高的遺傳多樣性水平。同一品種的不同樣品能較好地聚集在一起，但品種之間并未完全按形態(tài)學(xué)性狀聚類分支。構(gòu)建的指紋圖譜通過引物及引物組合能較好地將梔子品種進行區(qū)分。利用14對EST-SSR引物成功地構(gòu)建了10個梔子品種的指紋圖譜，研究結(jié)果可為梔子品種鑒定、親緣關(guān)系及品種起源提供科學(xué)依據(jù)。

梔子；EST-SSR；指紋圖譜；遺傳多樣性；親緣關(guān)系

梔子Ellis 為茜草科梔子屬常綠灌木，其花潔白如雪、香如凝脂，是我國著名的八大香花之一[1]，梔子果實還是大宗的中藥材原料，入藥具有瀉火除煩、清熱利濕、涼血散淤的功效，現(xiàn)代藥理研究發(fā)現(xiàn)梔子具有保肝、抗炎、鎮(zhèn)痛及改善心腦血管病等方面的作用[2]。梔子在長期的栽培利用過程中，使其習(xí)性、花、葉的形狀及大小、果實的形狀及大小等均發(fā)生了豐富的變異，在色素用、藥用和觀賞等不同利用方向都形成了穩(wěn)定的變異類型[3-4]，國內(nèi)外一些研究者命名了不同的變種或變型[5-6]，也有前人對梔子品種進行了系統(tǒng)的分類整理[7]，并從數(shù)量分類視角對梔子栽培品種與近緣種親緣關(guān)系進行了探討[8]。為規(guī)避環(huán)境因子和人為主觀意識等的干擾，有研究者采用分子標記法對梔子不同栽培類型進行鑒別以及研究它們之間的親緣關(guān)系，楊銳培[9]采用RAPD標記分析了江西樟樹梔子基地形態(tài)上區(qū)別明顯的14個桅子樣品，發(fā)現(xiàn)分子聚類結(jié)果和表型聚類結(jié)果類似；韓建萍等[10]采用AFLP標記分析了江西5個不同產(chǎn)地梔子的親緣關(guān)系，發(fā)現(xiàn)各地方樣品間遺傳變異明顯，且樣品間遺傳距離和地理距離相關(guān)性明顯。葛菲等[11-12]采用RAPD標記分析了梔子與雀舌梔子、重瓣梔子及水梔子間的親緣關(guān)系，結(jié)果顯示重瓣梔子與梔子遺傳距離最近，雀舌梔子與梔子遺傳距離最遠。魯耀邦等[13]采用RAPD標記分析了桅子與水桅子，野生桅子與栽培桅子間的DNA多態(tài)性。

SSR分子標記是以擴增片段的長度變異作為多態(tài)性的共顯性分子標記。由于微衛(wèi)星序列重復(fù)的長度及數(shù)目變化極其豐富，而使這種多態(tài)性檢測方法極為有效，目前已廣泛用于植物各水平遺傳多樣性、分子系統(tǒng)學(xué)、品種和純度鑒定、基因定位、遺傳圖譜構(gòu)建、比較基因組學(xué)等各方面研究中。在對植物種下等級親緣關(guān)系、種質(zhì)鑒定研究中，相對其它分子標記表現(xiàn)出明顯的優(yōu)越性[14-16]。本實驗對梔子常見主栽品種構(gòu)建了轉(zhuǎn)錄組微衛(wèi)星分子標記（transcriptome microsatellite markers，EST-SSR）指紋圖譜，以遺傳距離矩陣為分析對象，按不加權(quán)成組配對法（UPGMA）進行聚類分析，建立梔子品種間的親緣關(guān)系樹狀圖。系統(tǒng)探討梔子栽培品種遺傳距離和親緣關(guān)系，以期為梔子品種鑒定、親緣關(guān)系及品種起源提供科學(xué)依據(jù)。

1 材料與方法

1.1 材料

實驗材料為10個梔子品種[7]和1個梔子栽培原種共39份，每個品種1～5份樣本。所有實驗材料由江西省林業(yè)科學(xué)院鄧紹勇副研究員鑒定為梔子Ellis及其不同的品種類型，采集新鮮幼嫩葉片放入自封袋內(nèi)，隨即加入變色硅膠并搖勻（變色硅膠與樣品質(zhì)量比約為10∶1），封好袋后于常溫干燥處保存。根據(jù)硅膠變色情況及時加換硅膠。樣品來源等基本情況詳見表1。

表1 梔子品種取樣材料

Table 1 Information on ten cultivars of G.jasminoides

編號品種名稱品種來源地編號品種名稱品種來源地 1寬棱水梔1江西省樟樹市吳城藥材基地種植21金福水梔1湖北省宜昌市金福村栽培 2荷花梔子1廣東省廣州市園林栽培22金福水梔2湖北省宜昌市金福村栽培 3荷花梔子2廣東省廣州市園林栽培23金福水梔3湖北省宜昌市金福村栽培 4荷花梔子3廣東省廣州市園林栽培24太湖山梔1安徽省潛山太湖 5荷花梔子4廣東省廣州市園林栽培25太湖山梔2安徽省潛山太湖 6荷花梔子5廣東省廣州市園林栽培26山梔子1江西省永豐縣瑤里 7大花梔子1重慶市27山梔子2江西省永豐縣瑤里 8大花梔子2重慶市28山梔子3江西省永豐縣瑤里 9大花梔子3重慶市29山梔子4江西省永豐縣瑤里 10小白蟾1廣東省東莞市園林栽培30山梔子5江西省永豐縣瑤里 11小白蟾2廣東省東莞市園林栽培31球果梔子1江西省樟樹市隍城梔子GAP藥材基地種植 12小白蟾3廣東省東莞市園林栽培32球果梔子2江西省樟樹市隍城梔子GAP藥材基地種植 13雀舌梔子1湖北省武漢市園林栽培33白蟾1江西省南昌市園林栽培 14雀舌梔子1江西省南昌市園林栽培34白蟾2江西省南昌市園林栽培 15雀舌梔子2江西省南昌市園林栽培35白蟾3江西省南昌市園林栽培 16雀舌梔子3江西省南昌市園林栽培36小白蟾A1廣東省東莞市園林栽培 17水梔子1江西省樟樹市吳城藥材基地種植37小白蟾A2廣東省東莞市園林栽培 18水梔子2江西省樟樹市吳城藥材基地種植38小白蟾B1廣東省東莞市園林栽培 19水梔子3江西省樟樹市吳城藥材基地種植39小白蟾B2廣東省東莞市園林栽培 20水梔子4江西省樟樹市吳城藥材基地種植

1.2 方法

采用改進的CTAB裂解-硅珠吸附法從變色硅膠保存的葉片中提取總基因組DNA，從25對梔子EST-SSR多態(tài)引物[17]中篩選出14對適用于梔子品種擴增的多態(tài)引物（表2），所有的PCR擴增反應(yīng)程序由Eppendorf 5331梯度PCR擴增儀（Eppendorf，Hamburg，Germany）執(zhí)行。反應(yīng)體系為：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；反應(yīng)程序為：94 ℃預(yù)變性3 min；94 ℃變性30 s，63 ℃退火30 s（每循環(huán)降低1 ℃），72 ℃延伸30 s，共15個循環(huán)；94 ℃變性30 s，57 ℃退火30 s，72 ℃延伸30 s，共15個循環(huán)；72 ℃延伸10 min；10 ℃保持。PCR擴增產(chǎn)物用8%聚丙烯酰胺凝膠電泳檢測，采用銀染技術(shù)顯帶。

表2 14對梔子EST-SSR引物

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

位點引物序列(5'→3')重復(fù)單元片段大小/bp退火溫度/℃GenBank 登記號 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 數(shù)據(jù)分析方法

采用多人判讀比對方式對膠圖數(shù)據(jù)進行判讀，按條帶長度大小從大到小用A、B、C、D、E…進行編號，讀帶結(jié)果如AB、BC等，若某一個樣本只有一條帶，讀帶結(jié)果則為AA、BB等。同時統(tǒng)計各位點條帶的片段大小，方便數(shù)據(jù)數(shù)值轉(zhuǎn)換，條帶分析完畢之后把所有的結(jié)果根據(jù)軟件格式要求輸入相應(yīng)軟件進行實驗的數(shù)據(jù)處理與統(tǒng)計分析。利用POPGEN32軟件[18]計算觀察等位基因平均數(shù)（a）、有效等位基因數(shù)目（e）、觀察雜合度（o）、期望雜合度（e）、Shannon多樣性指數(shù)（Nei's genetic identity，）、Nei多樣性指數(shù)（ei）以及遺傳距離（genetic distance，D）。采用NTSYS-pc 2.10e 軟件[19]的UPGMA法利用N遺傳距離（1978）構(gòu)建系統(tǒng)聚類圖。

2 結(jié)果與分析

2.1 梔子品種遺傳多樣性分析

14對梔子EST-SSR引物均在供試梔子品種樣品中得到了較好的多態(tài)擴增，所用引物信息詳見表2。10個梔子品種和1個梔子栽培原種共39份材料，14對EST-SSR引物共檢測到62個等位基因，每個位點實際擴增N2～8個，引物eGJ15擴增等位基因最多為8個，平均為4.43；N為1.52～5.69，N為3.24。39個品種樣品的為1.23，N為0.65，觀測雜合度（）變化范圍為0.15～0.85，平均為0.51；期望雜合度（）變化范圍為0.35～0.83，平均為0.66（表3），結(jié)果顯示梔子品種有著較高的遺傳多樣性水平。引物eGJ19、eGJ26、eGJ134對梔子39份品種樣品的PCR擴增電泳檢測結(jié)果見圖1。

表3 梔子品種14個SSR位點的遺傳多樣度

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

基因樣本數(shù)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 梔子品種間遺傳關(guān)系分析

樣品間的遺傳距離在?0.118～2.859，平均為0.761；相似系數(shù)在0.057～1.125，平均為0.497（表4），大花梔子和山梔子間遺傳距離最大。以遺傳距離矩陣為分析對象，利用NTSYS-pc 2.1軟件按UPGMA法進行聚類分析，建立10個梔子品種和1個梔子栽培原種共39份材料間的親緣關(guān)系樹狀圖（圖2）。從聚類結(jié)果來看，除栽培梔子即山梔子5個樣本、金福水梔3個樣本及大花梔子未完全聚集在一起，尤其是金福水梔3個樣本聚類較為分散，其他8個品種各樣本均聚集在一起。其中寬棱水梔顯示和其他品種親緣關(guān)系最遠，荷花梔子顯示和太湖山梔、白蟾親緣關(guān)系較近，球果梔子顯示和山梔子親緣關(guān)系較近，小白蟾顯示和水梔子親緣關(guān)系較近，大花梔子、雀舌梔子聚類顯示則較為獨立。

圖1 部分SSR引物對梔子39份品種樣品的PCR擴增電泳檢測

表4 梔子品種間的D和I矩陣

Table 4 D and I matrices among Gardenia varieties

編號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****

對角線以上為，對角線以下為

is above diagonal andis below diagonal

圖2 10個梔子品種聚類圖

2.3 梔子品種SSR指紋圖譜構(gòu)建

根據(jù)多態(tài)引物擴增的電泳結(jié)果，采用在相同遷移率位置上有帶記為“1”，無帶記為“0”的讀帶方法，以14個SSR標記構(gòu)建梔子10個品種的指紋圖譜（表5），由表5可見，引物eGJ15和eGJ19多態(tài)性最為豐富，它們組合一起基本可以把10個梔子品種區(qū)分開，其他引物通過3個或3個以上組合一起也可以完成對梔子所有品種的區(qū)分。

3 討論

梔子在觀賞栽培和果用栽培方面都保留有豐富的品種資源[7]，對梔子品種進行科學(xué)規(guī)范的分類整理，研究掌握梔子品種遺傳多樣性和遺傳關(guān)系，明晰主栽梔子品種起源和系統(tǒng)進化關(guān)系，可以為梔子品種鑒定和梔子種質(zhì)資源創(chuàng)制提供科學(xué)依據(jù)。本實驗采用14對梔子EST-SSR引物對10個梔子品種和1個梔子栽培原種共39份材料進行擴增，共檢測到62個等位基因，39個品種樣品的為1.23，N為0.65，高于同樣栽培歷史悠久的觀賞海棠[16]和油茶[15]等木本植物，體現(xiàn)出梔子品種具有豐富的遺傳多樣性。在對梔子品種遺傳距離研究方面，葛菲等[11-12]采用RAPD標記分析顯示重瓣梔子（白蟾）與梔子遺傳距離最近，雀舌梔子與梔子遺傳距離最遠，本研究結(jié)論也顯示山梔子與白蟾遺傳距離相對山梔子與雀舌梔子遺傳距離要更近，且大花梔子和山梔子間遺傳距離最大。

楊銳培[9]采用RAPD標記分析了江西樟樹吳城鄉(xiāng)康成梔子基地形態(tài)上區(qū)別明顯的14個桅子樣品，14個樣品明顯區(qū)分成2組，發(fā)現(xiàn)分子聚類結(jié)果和表型聚類結(jié)果類似。以葉、花、果形態(tài)學(xué)性狀為數(shù)據(jù)基礎(chǔ)的數(shù)量分類視角對梔子品種進行聚類時，基本按色素用、藥用和觀賞用形成聚集分支[8]。而采用EST-SSR分子標記的梔子品種聚類并沒有得出類似的結(jié)果，但除了山梔子5個樣本、金福水梔3個樣本及大花梔子未完全聚集在一起，尤其是金福水梔3個樣本聚類較為分散，其他8個品種各樣本均聚集在一起。其中寬棱水梔顯示和其他品種親緣關(guān)系最遠，荷花梔子顯示和太湖山梔、白蟾親緣關(guān)系較近，球果梔子顯示和山梔子親緣關(guān)系較近，小白蟾顯示和水梔子親緣關(guān)系較近，大花梔子、雀舌梔子聚類顯示則較為獨立。

植物品種鑒定的可靠性受到多種情況的影響，如通過表型鑒定往往受鑒定者經(jīng)驗、材料完整程度、季節(jié)及材料特性等影響，而通過DNA指紋識別來完成品種鑒定可以克服上述一些因素的影響。采用SSR標記構(gòu)建植物品種指紋具備多態(tài)性高、帶型清晰、穩(wěn)定性和重復(fù)性好、操作簡單、成本低等優(yōu)點。文雁成等[20]通過比較研究認為SSR標記比SRAP標記更適合以引物組合法構(gòu)建品種指紋圖譜。因此，SSR標記構(gòu)建的指紋圖譜可作為梔子品種間區(qū)別的有力參考。本研究根據(jù)多態(tài)引物擴增的電泳結(jié)果，以14個SSR標記構(gòu)建梔子10個品種的指紋圖譜，通過引物指紋的兩兩組合，或者多個引物指紋的組合，可以達到區(qū)分品種的目前，其中引物eGJ15和eGJ19多態(tài)性最為豐富，它們組合一起基本可以把10個梔子品種區(qū)分開，使采用分子手段鑒定梔子品種成為現(xiàn)實。

表5 10個梔子品種14個SSR標記的指紋圖譜

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

編號品種中名標記代碼 eGJ4eGJ6eGJ11eGJ15eGJ19eGJ26eGJ41eGJ87eGJ91eGJ98eGJ123eGJ134eGJ144eGJ145 1寬棱水梔100010000100101000000000010001010000100110000100011001000011001 2荷花梔子100110100001000001000001100010010000100110000110010101010100110 3荷花梔子200110100001000010000001100010010000100110000110011001010100110 4荷花梔子300110100001001000000001100010010001001000010010011001010100110 5荷花梔子400110100001000010100001100010010000101000010010010100110100110 6荷花梔子500110100001000000100001100010010000101000010010010100110100110 7大花梔子100111010001000001000000000101010010010000010001000000100011001 8大花梔子201100100001000100000000100110000101001000010001010001000010101 9大花梔子300110010010000100000000100110011001001010010011000001011000011 10小白蟾100110001010100100010001010011010001010100010001011010001010001 11小白蟾200110001010110000010001010011011001010100010001011010001100001 12小白蟾300110001010101000001001010011011001010100010001011010001010001 13雀舌梔子100100110010001000010010100010010000100101010001010101000101010 14雀舌梔子100100110010000001000010100010010000100101000101010101000100101 15雀舌梔子200100110010000000010010100010010000100101001001010101000010101 16雀舌梔子300010010010000000001000100010010000100100010101010101000010101 17水梔子100101000110100100010000101010110001011000010101011000010010001 18水梔子200111000110100100000000010110010001011000010101011100010010001 19水梔子300101000110100100000000010110110010011000000101011100010010001 20水梔子400101000110100100000000010010110001011000100101010100010010001 21金福水梔100100000110000100000000100010100000110100001000010100000101010 22金福水梔201000101010000010000100100010010001000110001111000100101001000 23金福水梔311001010001000000100001100001011001010100001100110000101000110 24太湖山梔100110110001000001100010100010110001000110000110010101010100010 25太湖山梔200110100001000000100010100010100001000110000110010101010101100 26山梔子110100101001000010000000100010101011000101000110010101011000110 27山梔子210100101001000010000001000001101001000110000110010101011000110 28山梔子310100101001000010000001000010100001000110000110011100010100001 29山梔子400110101001000001000011000010010001001010000110011100011001010 30山梔子500100100001000100001001000010010011000110000101000100010100100 31球果梔子110100011010000010100000100011101001001000000100011100010100100 32球果梔子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

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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

國家自然科學(xué)基金資助項目（32060356）；國家自然科學(xué)基金資助項目（31760220）；中央財政林業(yè)科技推廣示范項目（JXTG[2022]12號）；江西省重點研發(fā)計劃項目（20203BBF63024）

鄧紹勇（1982－），男，博士，副研究員，主要研究方向為藥用植物資源開發(fā)與利用。Tel: (0791)83833641 E-mail: jxforestry@163.com

[責(zé)任編輯 時圣明]