常用中药百合为百合科植物卷丹 (Lilium Lancifolium)、百合 (Lilium brownie) 或细叶百合 (Lilium pumilum) 的肉质鳞叶。《中国药典》(2010版) 记载百合性甘、味寒,归心、肺经,具有养阴润肺、清心安神的功能。百合是集药用、食用和观赏为一体的经济植物,临床上主要用于治疗燥咳、肺结核、精神分裂症、更年期综合征、糖尿病等,应用广泛^[1]。

据《中国植物志》记载,百合属植物共100余 种,在中国主要分布约有39种,包括一些中国特有种,如毛百合 (Lilium dauricum)、新疆百合 (Lilium martagon)、台湾百合 (Lilium formosanum)、野百合 (Crotalaria sessiliflora)、岷江百合 (Lilium regale)、紫花百合 (Lilium souliei) 等。百合属中很多植物均为保健产品的重要原料,但百合属中丰富的种质资源给其鉴定及应用带来困难。由于百合属各物种形态特征相近,且存在很多变种,利用传统的形态、显微等特征很难准确快速的对其进行鉴别。通过性状、形态特征鉴别,应用的范围较小,而且鉴别需要一定的专业知识以及主观判断,没有统一的标准^{[2, 3]}。由于百合属中存在的活性成分的种类较多,运用化学方法进行鉴定的结果不能准确直观的反应药材真伪^[4]。早期的一些分子标记技术,如RAPD等,对DNA质量要求较高,结果不稳定,差异性主要表现在种内变异^{[5, 6]}。DNA条形码作为一种新的分子标记技术,操作简单,所需材料少,结果稳定可重复,通用性强,可以迅速、准确鉴定物种,且应用已十分广泛^{[7, 8, 9, 10, 11, 12]}。利用条形码技术对百合属植物的鉴别研究较少,且没有在整个属的水平进行系统的鉴别分析^[13]。本文通过实验,并结合GenBank数据库数据,分析ITS2、ITS、psbA-trnH、matK、rbcL五条常用条形码序列对百合属植物的鉴别能力,以筛选能鉴别百合属植物的优选序列及为百合属植物的分子鉴别提供依据。

实验所用材料百合、卷丹与细叶百合采于湖南、陕西、北京等地 (详见表 1)。经中国医学科学院药用植物研究所林余霖教授鉴定分别为百合 (Lilium brownie)、卷丹 (Lilium Lancifolium)、细叶百合 (Lilium pumilum),凭证标本保存于中国医学科学院药用植物研究所。实验所用试剂主要有天根植物 基因组DNA提取试剂盒; 2×Taq Mix; ddH₂O等。

表 1(Table 1)

Table 1 The source of samples for Lilium brownii and adulterants Experiment number Latin name Collection time Collection sites Accession number BH1 Lilium brownii 2012.6.9 SangZhi Hunan KJ161313 BH2 Lilium brownii 2012.7.6 DanFeng Shaanxi KJ161314 JD1 Lilium lancifolium 2012.6.9 SangZhi Hunan KJ161316 JD2 Lilium lancifolium 2012.7.6 DanFeng Shaanxi KJ161317 XY1 Lilium pumilum 2012.7.6 DanFeng Shaanxi KJ161323 XY2 Lilium pumilum 2012.7.8 ShangLuo Shaanxi KJ161319 XY3 Lilium pumilum 2012.7.9 HanZhong Shaanxi KJ161320 XY4 Lilium pumilum 2012.7.10 HanZhong Shaanxi KJ161321 XY5 Lilium pumilum 2012.7.29 MangShan Beijing KJ161322 DH Lilium leichtlinii 2012.7.5 ShangLuo Shaanxi KJ161315 SX Lilium regale 2012.7.16 HaiDian Beijing KJ161318

Table 1 The source of samples for Lilium brownii and adulterants

取干燥植物样品30 mg,球磨仪 (Retsch MM400,Germany) 1 800 r·min^-1研磨2 min (加入两粒灭菌小钢珠),使用天根植物基因组DNA提取试剂盒 (Tiangen Biotech Co.,China) 提取样 品总DNA。具体步骤如下,研磨后样品加入2.0 EP管中,加入缓冲液GP1 (65 ℃提前预热) 700 μL、巯基乙醇1 μL,65 ℃水浴1 h; 水浴后加入700 μL氯仿-异戊醇 (24∶1) 混合液,混匀后12 000 r·min^-1离心 5 min; 取上清液,加等体积异丙醇,混匀15～20 min; 分装过吸附柱,12 000 r·min^-1离心40 s; 弃废液,加缓冲液GD 500 μL,12 000 r·min^-1离心40 s; 弃废液,加漂洗液PW 700 μL,12 000 r·min^-1离心40 s; 弃废液,加漂洗液PW 500 μL,12 000 r·min^-1离心40 s; 弃废液,空管离心2 min,室温风干后加100 μL ddH₂O洗脱DNA,12000 r·min^-1离心2 min,即得样品总DNA模板,-20 ℃保存备用。

PCR体系总体积为25 μL,其中包含: 2×Taq PCR Mix 12.5 μL、DNA模板2 μL、ddH₂O 8.5 μL、引物ITS 6F/4R各1 μL。引物序列 及PCR反应条件见表 2。扩增结束后以1% 琼脂糖凝胶电泳检测,凝胶成像分析系统分析。

表 2(Table 2)

Table 2 The primer sequence and PCR reaction condition Primer name Primer sequences (5'-3') PCR reaction condition ITS2 2F ATGCGATACTTGGTGTGAAT 94 ℃ 5 min; 3R GACGCTTCTCCAGACTACAAT 94 ℃ 30 s,56 ℃ 30 s,72 ℃ 45 s,40 cycles; 72 ℃ 10 min; ITS 4R TCCTCCGCTTATTGATATGC 94 ℃ 5 min; 5F GGAAGTAAAAGTCGTAACAAGG 94 ℃ 1 min,50 ℃ 1 min,72 ℃ 1.5 min+3 s/cycle,30 cycles; 72 ℃ 7 min; psbA-trnH fwd PA GTTATGCATGAACGTAATGCTC 94 ℃ 4 min; rev TH CGCGCATGGTGGATTCACAATCC 94 ℃ 30 s,55 ℃ 1 min,72 ℃ 1 min,35 cycles; 72 ℃ 10 min; matK 3F_KIM CGTACAGTACTTTTGTGTTTACGAG 94 ℃ 1 min; 1R_KIM ACCCAGTCCATCTGGAAATCTTGGTTC 94 ℃ 30 s,52 ℃ 20 s,72 ℃ 50 s,35 cycles; 72 ℃ 5 min; rbcL 1f ATGTCACCACAAACAGAAAC 95 ℃ 4 min; 724r TCGCATGTACCTGCAGTAGC 94 ℃ 30 s,55 ℃ 1 min,72 ℃ 1 min,35 cycles; 72 ℃ 10 min;

Table 2 The primer sequence and PCR reaction condition

PCR产物直 接使用ABI3730XL测序仪 (Applied Biosystems Co.,USA) 进行双向测序。序列拼接软件CodonCode Aligner (V3.7.1) 对测序峰图文件进行拼接,去除引物区及低质量序列; 使用Clustal (MEGA 5.0) 对拼接后序列进行比对分析; 同时本研究从GenBank数据库中搜集并下载百合属ITS2、ITS、psbA-trnH、rbcL、matK序列717条(表 3),整合处理后结合样品序列,运用MEGA 5.0建立NJ树,分析百合及其伪品亲缘关系; 采用Perl语言统计不同序列种内、种间遗传差异、barcoding gap; 运用相似性搜索算法 (BLAST1) 和最近距离法 (K2P nearest distance) 考察各序列的鉴定成功率^[14]。

表 3(Table 3)

Table 3 Accession numbers of GenBank database Barcode Species Accession number ITS Lilium amoenum AB035284 Lilium brownie AB035282 EU303289 AF088199 HQ692122 HQ692121 HQ692120 HQ692119 Q692118 HQ692117 HQ692116 HQ448858 EU303282 Lilium davidii AF088195 EU303284 HQ692078 HQ692077 HQ692076 HQ692075 HQ692074 HQ692073 HQ692072 HQ692071 HQ692070 HQ692069 HM045426 AB020461 AB020426 Lilium lancifolium AF088193 AF074472 HQ692094 HQ692093 HQ692092 HQ686070 HQ724825 HQ724824 AB020456 AB020453 Lilium bakerianum HQ692143 HQ692142 HQ692141 HQ692140 HQ456829 HM045468 HM045428 EU296433 Lilium callosum AF074468 HQ686065 HQ223061 HQ223060 HQ223059 HQ223058 HQ223057 HQ223056 HQ223055 HQ724817 AB020471 HQ223062 HQ686066 Lilium cernuum AF088196 HQ686064 HM045427 HQ223067 HQ223066 HQ223065 HQ223063 HQ223064 HQ724818 AB020463 Lilium concolor AF074469 JN785965 JN785964 JN785963 HQ686067 HM045460 HQ223070 HQ223069 HQ223068 HQ724820 HQ724819 EU303283 AB020441 AB020423 Lilium dauricum AF074470 HQ724821 HM045446 EU303290 AB020473 Lilium pensylvanicum JN785968 JN785967 JN785966 HQ686069 Lilium distichum JN785971 JN785970 JN785969 HQ686075 HM045451 HQ223042 HQ223041 HQ223040 HQ223039 HQ724822 EU303295 Lilium duchartrei AF088208 HQ692068 HQ692067 HQ692066 HQ692065 HQ692061 HQ692060 HQ448864 HM045429 AB035283 Lilium fargesii JN785974 JN785973 JN785972 HM045459 Lilium henricii JN785977 JN785976 JN785975 AF088212 AB035285 HM045456 Lilium henryi AF088217 HM045462 EU303292 AB020467 Lilium leucanthum EU303286 HQ692130 HQ692129 HQ692128 HQ448861 HM045466 HM045463 AB020443 Lilium longiflorum AF074474 HQ686076 AY684927 AB020458 Lilium lophophorum AY616747 HQ692106 HQ692105 HQ692104 HQ692103 HQ692102 HQ692101 HQ692100 HQ692099 HQ692098 HQ692097 HQ692096 HQ692095 HM045455 Lilium martagon AF088203 FJ979912 FJ979911 FJ979910 FJ979909 FJ979908 FJ979907 FJ979906 EF042777 AB020455 HM045452 Lilium nanum AF088210 HQ687289 AB020446 JN785980 JN785979 JN785978 HM045458 Lilium papilliferum HQ687262 Lilium rosthornii HQ687261 EU303297 Lilium sargentiae AF088215 HQ692114 HQ692115 HQ692112 HQ687258 HQ456832 EU303291 AB020425 Lilium sempervivoideum HM045467 Lilium souliei JN785983 JN785982 JN785981 JQ724631 Lilium speciosum AF074479 EU303293 HM045461 AB020431 Lilium sulphureum AF088213 HQ692127 HQ692126 HQ692125 HQ692124 HQ692123 HQ448862 HM045464 EU219391 Lilium taliense HQ692111 HQ692110 HQ692109 HQ692108 HQ692107 HM045425 AB020421 Lilium tsingtauense AF088205 HQ687259 HQ686073 HQ223053 HQ223052 HQ223050 HQ223051 HQ223049 HQ223048 HQ724827 EU303296 AB020433 HQ686074 Lilium wardii JN785986 JN785985 JN785984 AF088206 AB035287 Lilium xanthellum HQ692154 HQ692153 HQ692152 HQ692151 HQ687294 Continued Lilium leichtlinii HQ223077 AF074473 HQ686072 HQ724826 AB020454 Lilium nepalense AF088198 HQ687293 AB020444 Lilium pumilum AF088194 HQ692088 HQ692087 HQ692086 HQ692085 HQ692084 HQ692083 HQ692082 HQ692081 HQ692080 HQ692079 HQ686071 JF778866 HQ456830 HM045445 GU565939 EU303287 AB020430 Lilium regale AF088214 HQ692091 HQ692090 HQ692089 HQ456831 EU219390 AB020434 ITS2 Lilium lancifolium JF778864 Lilium pumilum JF778871 matK Lilium amoenum AB049515 Lilium brownii AB049517 JQ724604 HQ692223 HQ692222 HQ692221 HQ692220 HQ692219 HQ692218 HQ692217 Lilium davidii AB049503 JQ724606 AY624460 HQ692179 HQ692178 HQ692177 HQ692176 HQ692175 HQ692174 HQ692173 HQ692172 HQ692171 HQ692170 Lilium bakerianum JQ724602 HQ692244 HQ692243 HQ692242 HQ692241 JQ724601 HQ687300 Lilium callosum JF491157 JF491156 JF491155 JF491153 JF491154 JF491152 JF491151 JF491158 Lilium cernuum AB049506 JQ724595 JF491163 JF491162 JF491161 JF491160 JF491159 JN417330 JF491165 JF491164 Lilium concolor JN785995 JN785994 JN785993 JQ724605 JF491169 JF491168 JF491167 JF491166 JN417331 GQ434067 AB049511 Lilium dauricum AB049497 Lilium pensylvanicum JN785998 JN785997 JN785996 JN417332 Lilium distichum JN786001 JN786000 JN785999 JQ724594 JX903654 JF491139 JF491138 JF491137 JN417333 Lilium duchartrei JQ724578 HQ692169 HQ692168 HQ692167 HQ692166 HQ692165 HQ692163 HQ692164 HQ692162 HQ692161 HQ692160 HQ692159 Lilium fargesii JN786007 JN786005 JN786006 JQ724582 HQ687301 Lilium henricii JN786004 JN786002 JN786003 JQ724597 HQ687305 Lilium henryi AB049505 JQ724583 Lilium leucanthum JQ724580 JQ724579 HQ692231 HQ692230 HQ692229 Lilium longiflorum AB049525 Lilium lophophorum JQ724610 AY624462 HQ692206 HQ692207 HQ692205 HQ692204 HQ692203 HQ692202 HQ692201 HQ692200 HQ692199 HQ692198 HQ692197 HQ692196 HQ687303 Lilium martagon JQ724596 Lilium nanum AB049509 JQ724598 HQ687295 JN786010 JN786009 JN786008 Lilium sargentiae JQ724576 HQ692216 HQ692214 HQ692213 HQ692215 Lilium sempervivoideum JQ724612 Lilium souliei JN786013 JN786012 JN786011 Lilium speciosum AB049526 AY624467 JQ724613 AB049504 Lilium sulphureum JQ724614 HQ692228 HQ692227 HQ692226 HQ692225 HQ692224 Lilium taliense AB049492 JQ724590 HQ692212 HQ692211 HQ692210 HQ692209 HQ692208 Lilium tsingtauense JQ724599 JX903656 JF491149 JF491148 JF491147 JF491146 JF491145 JF491144 JN417337 Lilium lancifolium JF491174 JF491173 JF491172 JF491171 JF491170 HQ692195 HQ692194 HQ692193 JF972949 JQ724577 Lilium leichtlinii JF491175 JN417335 Lilium nepalense AB049493 JQ724585 HQ687299 Lilium wardii JN786016 JN786015 JN786014 Lilium xanthellum JQ724588 HQ692255 HQ692254 HQ692253 HQ692252 HQ687304 JQ724589 Lilium pumilum JQ724592 HQ692189 HQ692188 HQ692186 HQ692187 HQ692185 HQ692184 HQ692183 HQ692182 HQ692181 HQ692180 GQ434062 Lilium regale AB049491 JQ724624 HQ692192 HQ692191 HQ692190 JN417338 AY624464 AB040200 psbA-trnH Lilium bakerianum HQ692443 HQ692442 HQ692441 HQ692440 HQ687263 HM053710 Lilium brownie GU445359 HQ692422 HQ692421 HQ692420 HQ692419 HQ692417 HQ692418 HQ692416 JF708209 HQ687264 Lilium_davidii HQ687265 HQ692378 HQ692377 HQ692376 HQ692375 HQ692374 HQ692373 HQ692372 HQ692371 HQ692370 HQ692369 Lilium lancifolium HQ692394 HQ692393 HQ692392 HQ687277 Lilium cernuum HM053712 Lilium pumilum HQ692388 HQ692387 HQ692386 HQ692385 HQ692384 HQ692383 HQ692382 HQ692381 HQ692380 HQ692379 HM053722 HM053721 HM053720 HM053719 HM053718 HM053717 HM053716 HM053715 HM053714 HM053713 GQ434916 HQ687273 Continued Lilium regale HQ692391 HQ692390 HQ692389 HQ687274 Lilium concolor JN786025 JN786023 JN786024 HQ687281 GQ434923 Lilium distichum JN786031 JN786030 JN786029 HM053711 Lilium duchartrei HQ692368 HQ692367 HQ692366 HQ692365 HQ692364 HQ692363 HQ692362 HQ692361 HQ692360 HQ692359 HQ692358 HQ687266 Lilium fargesii JN786034 JN786033 JN786032 HQ687283 Lilium formosanum GU445360 Lilium henricii JN786037 JN786036 JN786035 HQ687284 Lilium henryi HQ687285 Lilium leucanthum HQ692430 HQ692429 HQ692428 HQ687269 HM053730 Lilium lophophorum HQ692406 HQ692405 HQ692404 HQ692403 HQ692402 HQ692401 HQ692400 HQ692399 HQ692398 HQ692396 HQ692395 HQ687268 Lilium martagon HQ687270 Lilium nanum JN786040 JN786039 JN786038 Lilium pensylvanicum JN786028 JN786027 JN786026 HQ687282 Lilium sargentiae HQ692415 HQ692414 HQ692413 HQ692412 HQ687275 Lilium souliei JN786043 JN786041 JN786042 Lilium speciosum HM053731 Lilium sulphureum HQ692427 HQ692426 HQ692425 HQ692424 HQ692423 HQ687276 Lilium taliense HQ692411 HQ692410 HQ692409 HQ692408 HQ692407 HM053709 Lilium tsingtauense HQ687287 Lilium wardii JN786046 JN786045 JN786044 Lilium xanthellum HQ692454 HQ692453 HQ692452 HQ692451 rbcL Lilium bakerianum AB034923 HQ692343 HQ692342 HQ692341 HQ692340 HQ687318 Lilium brownii HQ692322 HQ692321 HQ692320 HQ692319 HQ692318 HQ692317 HQ692316 Lilium davidii GQ436288 HQ692279 HQ692278 HQ692277 HQ692276 HQ692275 HQ692274 HQ692273 HQ692272 HQ692271 HQ692270 Lilium lancifolium AB034937 HQ692295 HQ692294 HQ692293 JF972917 Lilium cernuum JN417419 Lilium concolor JN417420 GQ436311 Lilium distichum JX903238 JN417422 Lilium duchartrei HQ692269 HQ692268 HQ692266 HQ692267 HQ692265 HQ692264 HQ692263 HQ692262 HQ692261 HQ692260 HQ692259 Lilium fargesii HQ687319 Lilium formosanum AB034933 Lilium henricii HQ687323 Lilium henryi AB034924 Lilium leucanthum HQ692330 HQ692329 HQ692328 Lilium lophophorum HQ692306 HQ692305 HQ692304 HQ692303 HQ692302 HQ692301 HQ692300 HQ692299 HQ692298 HQ692297 HQ692296 Lilium nanum HQ687313 Lilium pensylvanicum JN417421 Lilium sargentiae HQ692315 HQ692314 HQ692313 HQ692312 Lilium speciosum AB034922 Lilium sulphureum HQ692327 HQ692326 HQ692325 HQ692324 HQ692323 Lilium taliense HQ692311 HQ692310 HQ692309 HQ692308 HQ692307 Lilium tsingtauense JX903240 JN417426 Lilium xanthellum HQ692354 HQ692353 HQ692352 HQ692351 HQ687322 Lilium leichtlinii AB034932 JN417424 Lilium nepalense HQ687317 Lilium pumilum HQ692289 HQ692288 HQ692287 HQ692286 HQ692285 HQ692284 HQ692283 HQ692282 HQ692281 HQ692280 GQ436305 Lilium regale HQ692292 HQ692291 HQ692290 AB088816

Table 3 Accession numbers of GenBank database

各序列数量、GC含量、遗传距离、鉴定效率等信息见表 4。由数据可以看出,ITS2序列数据库数量较少,只有2条,加上从ITS序列中注释提取出的ITS2序列共计253条。ITS序列数量最多,为275条(包括实验数据),rbcL序列数量最少,为105条。各序列GC含量差异较大,ITS2序列最高,平均含量 达到64.85%,psbA-trnH与matK序列GC含量较低; 平均遗传距离psbA-trnH序列最大,为0.311 8,其次为ITS2,为0.056 3,rbcL序列最小。BLAST1方法分析各序列的鉴定效率,ITS序列比其他4条均较高,为58.91%,rbcL序列最小,为7.41%; 最近距离法分析结果可看出,ITS序列比其他序列均较高,为62.55%,rbcL序列最低,为12.96%。综合分析,ITS序列可作为鉴定百合属的优选序列。

表 4(Table 4)

Table 4 Sequence information and identification efficiency Maker ITS2 ITS psbA-trnH matK rbcL Number 253 275 140 205 105 GC content/% 64.85 60.90 31.24 30.86 43.31 Genetic distance Max 0.232 9 0.267 0 2.202 4 0.981 1 0.014 9 Min 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 Average 0.056 3 0.052 7 0.311 8 0.086 9 0.003 1 Identification efficiency BLAST1/% 37.94 58.91 19.29 13.66 7.41 Nearest distance/% 60.08 62.55 25.71 32.68 12.96

Table 4 Sequence information and identification efficiency

种内种间差异是分析条形码序列的重要指标。合适的条形码序列具有较大的种间变异、较小的种内 变异,以便准确鉴别同属不同种的物种。本研究从种间变异、平均种间变异、种间最小变异、种内变异、平均种内变异、种内最大变异分析不同序列的种内 种间差异 (表 5)。5条序列中,种间最小变异ITS2 序列最大 (0.014 2),其次为ITS序列 (0.012 9),最 小为rbcL (0.000 8); 种内最大变异psbA-trnH最大(0.589 2),其次为matK序列(0.139 7),最小为rbcL (0.001 3)。matK序列平均种间变异 (0.086 1) 小于平均种内变异 (0.115 8),其余ITS等4条序列均为平 均种间变异大于平均种内变异; rbcL序列虽然有较小的种内变异,但其种间变异同样较小; psbA-trnH序列虽然与ITS、ITS2序列的种间最小变异均小于种内最大变异,但psbA-trnH序列较ITS与ITS2序列,种间最小变异最小,而种内最大变异最大,且psbA-trnH序列种内变异均大于ITS与ITS2序列; ITS与ITS2序列种内种间差异相差不大,ITS序列较ITS2种内 差异较小,但ITS序列种间变异略小于ITS2。综合 分析,ITS序列是鉴别百合属较理想的条形码序列。

表 5(Table 5)

Table 5 Analysis of intra and inter-specific variation of different barcodes Marker ITS2 ITS psbA-trnH matK rbcL Theta 0.015 3 ± 0.013 3 0.016 4 ± 0.014 7 0.195 6 ± 0.188 5 0.043 3 ± 0.108 3 0.000 5 ± 0.000 6 Coalescent depth 0.040 3 ± 0.030 5 0.042 1 ± 0.035 4 0.589 2 ± 0.470 0 0.139 7 ± 0.341 9 0.001 3 ± 0.002 0 All intraspecific distance 0.014 3 ± 0.020 5 0.013 7 ± 0.024 6 0.151 7 ± 0.343 7 0.115 8 ± 0.310 8 0.000 6 ± 0.001 6 Theta prime 0.054 9 ± 0.022 2 0.050 0 ± 0.016 3 0.250 1 ± 0.222 3 0.057 1 ± 0.056 7 0.003 2 ± 0.001 8 Minimum interspecific distance 0.014 2 ± 0.023 2 0.012 9 ± 0.015 6 0.001 5 ± 0.002 9 0.001 2 ± 0.002 1 0.000 8 ± 0.001 9 All inter-specific distance 0.057 8 ± 0.028 0 0.054 3 ± 0.026 6 0.201 1 ± 0.374 4 0.086 1 ± 0.261 9 0.003 3 ± 0.002 3

Table 5 Analysis of intra and inter-specific variation of different barcodes

Barcoding gap是指序列种内与种间遗传变异由于显著差异而形成的间隔区^{[15, 16]}。如图 1为5条序列的barcoding gap图。5条序列均无明显的间隔区,rbcL序列种内种间变异交叉相对较少,但区分并不明显。相对其他序列,ITS序列虽然无明显间隔区,但种内与种间变异明显分为两大部分,其分布较高的遗传变异可以明显区分,因此在barcoding gap分析方面,ITS序列为鉴别百合属的较理想条形码序列。

Figure 1 Relative distribution of inter-specific divergence and intra-specific variation of different barcodes. A: ITS2; B: ITS; C: psbA-trnH; D: matK; E: rbcL

NJ树是基于K2P遗传距离建立,分析不同物种间的亲缘关系远近,以鉴别物种。该方法可直观展示不同物种的系统关系。如图 2所示,5条序列可以很好的将百合属不同物种分开。ITS序列可以将不同物种分到不同的分支上,与其他序列相比,能更好的区分百合属相近的物种,是鉴别百合属的优选序列。

Figure 2 Analysis of NJ tree built on different barcodes. A: ITS; B: psbA-trnH; C: rbcL; D: matK; E: ITS2

百合属原产于北半球的温带和寒带地区。我国是百合植物自然分布中心,但对于百合属植物资源的系统进化关系及相互间亲缘关系研究较少,导致我国在百合育种方面非常薄弱,大部分的商业栽培品种主要依赖于国外进口。在对百合属种质资源关系的研究中,前期多采用数量性状等对百合属进行分类研究^[17],但选用不同的器官性状分类差异可能较大,相对来说生殖器官性状比营养器官性状分类分析结果差异较小; 部分研究利用等位酶系统基因位点的等位基因等对百合属进行分类^{[18, 19]}; RAPD技术是研究百合分类较多的分子标记技术^{[20, 21, 22]},可以根据随机引物扩增出的片段的多态性进行鉴别,灵敏度较高,但结果不稳定,可重复性不强,适合种内不同个体间的鉴别。

DNA条形码是一种较新的分子标记技术,操作简便、技术成熟、通用性强,对于不同物种之间的鉴别能力较强,已有广泛的研究和应用基础。本研究运用ITS2、ITS、psbA-trnH、matK、rbcL 5条常用序列,从种内种间变异、barcoding gap、鉴定成功率3个方面分析DNA条形码对于百合属物种的鉴别能力,目的在于找到能快速、通用地鉴别百合属的DNA条形码序列。研究结果表明matK序列平均中间变异小于平均种内变异,说明matK序列有利于鉴别种内变异较大的物种,不适合在百合属水平上的鉴别; rbcL序列种内与种间变异均较小,可作为鉴别百合属的补充序列; psbA-trnH序列相对于ITS和ITS2序列种间差异小,而种内变异较大,因此不适合鉴别百合属物种; ITS2序列包含于ITS序列中,ITS序列较ITS2种内差异较小,但ITS序列种间变异略小于ITS2,且在实际鉴定工作中,获得ITS序列即可获得ITS2序列。虽然ITS序列在本实验中的鉴定成功率并不高,但考虑到GenBank数据库数据的复杂性及其来源的多样性,且在本实验中ITS序列在扩增效率、鉴定成功率、种内与种间变异、barcoding gap、系统发育树等方面均较其他序列表现良好,因此可以作为鉴别百合属的优选DNA条形码序列。本研究为百合属物种的鉴别提供新的分子鉴别方法,并为亲缘关系较复杂的属筛选理想DNA条形码鉴别序列提供理论 依据。