Research advances on molecular breeding technique of orchid
-
摘要:
兰花具有很高的观赏、药用、食用、生态和文化价值，因其丰富的种类、独特多样的花型、迷人的花色、怡人的香气和神秘的起源受到各国人民的喜爱。分子育种是兰花育种的发展方向，本文对近年来兰花基因组测序、主要育种目标性状的分子遗传基础和基因克隆以及转基因、基因编辑、分子标记辅助选择育种技术等方面的研究进展进行了综述，并对未来兰花分子育种技术研究的重点和品种创新进行了展望。
Abstract:Orchid has high ornamental, medicinal, edible, ecological and culture values, which is loved by people all over the world due to its plentiful species, diversified exquisite floral morphology, charming colour, pleasant aroma and mysterious origin. Molecular breeding is the developmental direction in orchid breeding and great progresses have been made in recent years. In this paper, we summarized the advancements on genome sequencing, molecular genetic basis and gene cloning of main breeding objective traits, the techniques of genetic transformation, gene editing and molecular marker assisted selection in orchid, and discussed the key points of future study on molecular breeding technique and variety innovation in orchid.
-
Keywords:
- Orchid /
- Breeding /
- Marker assisted selection /
- Genetic transformation /
- Gene editing
-
兰花是兰科Orchidaceae植物的总称，但在中国，兰花是指兰科兰属Cymbidium植物，特别是其中的地生种类，也就是今天的国兰[1]。兰科是开花植物中最大的科之一，已鉴定的兰科植物有899属29 199种，约占开花植物的8%~10%，除极地和极端干旱的沙漠外，世界各地均有兰花分布[2-3]。兰花具有很高的观赏价值、药用价值、食用价值、生态价值和文化价值，是世界著名的观赏花卉，也是中国传统十大名花之一[3]。作为观赏花卉，兰花既可以做切花、也可以做盆花，既可以观花、也可以赏叶，同时也是园林造景优良植材，深受世界各国人民的喜爱。作为进化程度最高、种类最丰富的植物，兰花也是当今生物学领域研究生命和进化的理想模式植物。新品种选育是兰花产业自主和高质量发展的基础，而育种方法是快速高效选育兰花优良品种的关键。目前，兰花育种的主要方法有引种驯化、选择育种、杂交育种、诱变育种和多倍体育种等[4-5]。这些育种方法虽然有效，但可利用资源范围窄、育种周期长，不能定向培育市场所需品种。分子育种是采用转基因或基因编辑技术创造变异、或通过分子标记辅助选择技术培育新品种的方法，主要包括转基因育种、基因编辑育种和分子标记辅助选择育种。近年来，随着兰花组学技术的发展，兰花主要育种目标性状的分子遗传基础、基因鉴定和克隆、高密度分子图谱构建等取得巨大进步，兰花转基因技术和分子标记辅助选择技术也取得明显进展，本文对此进行综述并对未来兰花分子育种技术研究的重点和品种创新进行了展望，为进一步推动兰花分子育种技术研究和新品种选育提供参考。
1. 兰花基因组测序和功能基因鉴定
1.1 基因组测序
自2015年Cai等[6]完成小兰屿蝴蝶兰Phalaenopsis equestris 基因组测序以来，迄今有来自4亚科8属18种共27份兰花完成了基因组测序，包括附生、地生和腐生3类(表1)[6-31]。其中，广东舌唇兰Platanthera guangdongensis基因组最大(4.27 Gb)[20]，深圳拟兰Apostasia shenzhenica基因组最小(348.73 Mb)[30]，天麻Gastrodia elata预测基因数最少(17 895个)[25]，染色体数最少的香荚兰Vanilla fragrans预测基因数最多(59 128个)[29]。基于基因组测序结果，对兰花叶艺、花型、花色、花期、适应性等性状的分子遗传基础分析结果表明，CH1基因家族显著缩减可能是导致墨兰Cymbidium sinense叶艺形成的重要原因[7]；光合天线和代谢途径相关基因表达水平降低引起建兰C. ensifolium叶艺的形成，叶片中与花发育相关的MADS-box基因表达水平显著提高导致花被状叶的形成[8]。
表 1 兰花基因组测序Table 1. Genome sequencing of orchid物种
Species染色体数
No. of
chromosomes基因组
大小/Gb
Genome size预测基因数
No. of
predicted genes重复序列/%
Repetitive
sequence测序技术及文献
Sequencing technique
and reference墨兰 Cymbidium sinense 20 3.52 29 638 77.78 Illumina HiSeq 2000[7] 建兰 C. ensifolium 20 3.62 29 073 80.58 Illumina HiSeq 2500 PacBio[8] 春兰 C. goeringii 20 3.99 29 556 88.87 Illumina Hiseq 2500[9] 春兰 C. goeringii 20 4.10 29 272 77.65 Illumina HiSeq 2500 PacBio[10] 硬叶兰 C. mannii 20 2.88 27 192 82.80 MGISEQ-2000[11] 铁皮石斛
Dendrobium officinale19 1.35 35 567 63.33 Illumina HiSeq 2000 PacBio[12] 铁皮石斛 D. catenatum 19 1.12 29 257 78.10 Illumina Hiseq2000[13] 鼓槌石斛 D. chrysotoxum 19 1.37 30 044 62.81 MGISEQ-2000 NovaSeqPacBio[14] 铁皮石斛 D. officinale 19 1.23 25 894 76.77 Illumina Hiseq 4000 PacBio[15] 霍山石斛 D. huoshanense 19 1.285 21 070 74.92 Illumina HiSeq 2000 PacBio[16] 金钗石斛 D.nobile 19 1.16 29 476 61.07 Illumina Novaseq6000[17] 小兰屿蝴蝶兰
Phalaenopsis equestris19 1.13 29 545 61.53 Illumina HiSeq 2000[6] 蝴蝶兰‘KHM190’
P. ‘KHM190’19 3.10 41 153 59.74 Illumina HiSeq 2000[18] 台湾蝴蝶兰 P. aphrodite 19 1.03 28 902 60.30 Illumina Hiseq 2000[19] 紫金舌唇兰
Platanthera zijinensis21 4.15 24 513 77.38 Illumina Hiseq 4000 PacBio[20] 广东舌唇兰
Platanthera guangdongensis21 4.27 22 559 82.18 Illumina Hiseq 4000 PacBio[20] 天麻 Gastrodia elata 18 1.06 18 969 66.18 Illumina HiSeq 2500[21] 天麻 G. elata 18 1.12 24 484 68.34 Illumina HiSeq 2000[22] 天麻 G. elata 18 1.04 21 115 66.36 PacBio Sequel II[23] 天麻 G. elata 18 1.05 18 844 74.92 Illumina NovaSeq 6000 PacBio[24] 天麻 G. elata 18 1.09 17 895 63.89 Illumina HiSeq 4000[25] 勐海天麻 G. menghaiensis 18 0.99 17 948 65.08 Illumina HiSeq 2500[26] 白及 Bletilla striata 18 2.37/2.43 26 673 PacBio[27] 香荚兰 Vanilla planifolia 14 1.5 29 167 44.30 Illumina HiSeq 2000[28] 香荚兰 V. planifolia 14 3.4 59 128 72.00 Illumina HiSeq 2000[29] 深圳拟兰
Apostasia shenzhenica34 0.35 21 841 42.05 Illumina HiSeq 2000 PacBio[30] 多枝拟兰 A. ramifera 34 0.37 22 841 44.99 Illumina Hiseq 2000[31] MADS-box基因影响花发育和形态。通过测序分析，在多枝拟兰A. ramifera、深圳拟兰、天麻、紫金舌唇兰Platanthera japonica、广东舌唇兰、香荚兰、小兰屿蝴蝶兰、台湾蝴蝶兰P. aphrodite、鼓槌石斛Dendrobium chrysotoxum、铁皮石斛D. catenatum、春兰C. goeringii 、建兰和墨兰中分别鉴定出30、36、40、43、43、55、51、56、58、63、69、71和94个MADS-box基因[2, 7, 9, 19]，且多数兰花中没有FLC、AGL12和AGL15基因。在小兰屿蝴蝶兰中有20个基因优先在花组织中表达，MIKC等5个基因只在花中表达，C/D类和B类基因AP3、AGL6基因家族的扩张和分化可能是兰花高度特化花型形成的原因[6]。CsSEP4基因正调控墨兰合蕊柱发育 [7]。C类基因CeAG-1和 CeAG-2在建兰花瓣中的高表达形成鼻状花瓣，在花芽中低表达时，则花中没有鼻，取而代之的是几轮花被片，形成具有多花被片分枝花序的花；CeSEP-2基因表达上调对兰花形成特化的唇瓣十分重要，下调则形成菊瓣花；唇瓣结构由CeAP3-1、CeAP3-2、CeAP3-3、CeAP3-4和CeAGL6-2基因控制，CeAP3-3、CeAP3-4和CeAGL6-2在萼片和花瓣中高表达则分别形成唇瓣状萼片和花瓣突变体[8]。
萜类化合物在兰花香气和抗逆性中起关键作用。在深圳拟兰、小兰屿蝴蝶兰、鼓槌石斛、金钗石斛D. nobile和墨兰中鉴定出TPS基因数分别为14、21、48、51和59个[6,7,14,17,30]。墨兰的花香与TPS基因家族扩张有关，与无香的墨兰相比，有香的墨兰TPS家族基因表达量更高[7]。单萜和倍半萜主要在花发育晚期和开花期形成，与其生物合成的相关基因GDPS、FDPS和LIS高表达有关，FDPS和GDPS主要在萼片、花瓣和唇瓣中表达，说明萜类化合物主要在花被中合成[8]。
花色是兰花主要育种目标性状。通过基因组测序和表达分析，在墨兰中鉴定出56个花青素合成代谢相关基因和125个MYBs，花青素合成代谢相关基因在绿色、黄色、粉色和紫黑色花中差异表达，其中，4个F3′H基因在红色和紫色花中高表达。调控花青素合成途径上游的MYBs一般在开紫黑色花的墨兰中表达上调，而调控下游的平行基因表达谱存在显著差异，此外，MYBs通过调控F3′5′H的表达影响墨兰的花色[7]。在白色和粉色台湾蝴蝶兰中，F3H的表达模式明显不同，R2R3-MYB在不同颜色蝴蝶兰中表达模式也不同[19]。
次生代谢物是药用兰花的主要育种目标性状。测序分析结果表明，铁皮石斛中SPS和SuSy基因扩张与石斛多糖形成有关[12]；甲基赤藓糖醇磷酸途径中DXS、LUT1、LUT2、LCY1、LUT5等相关基因的丢失，导致天麻中叶黄素、胡萝卜素和脱落酸等物质的缺乏[23]；OMT基因在秋水仙素生物合成中起重要作用[24]。
其他性状研究结果表明，墨兰CsSVP基因与其互作蛋白CsAP1和CsSOC1共同调控低温诱导开花[7]。附生兰耐热性与SDD1、PPP7和3MAT基因家族扩张有关[12]。鼓槌石斛花期短是由于在开花至凋谢过程中类胡萝卜素含量逐渐增加、叶黄素含量逐渐减少，导致ABA含量减少、乙烯含量增加所致[14]。
1.2 功能基因鉴定
功能基因鉴定是兰花分子育种的基础。迄今采用转基因、RNAi和VIGS等技术已从兰属、石斛属、蝴蝶兰属和文心兰属Oncidium兰花中鉴定出功能基因91个[32-103]。其中，通过将基因转入兰花中并验证功能的有24个，占比24.7%。已鉴定出功能的基因包括植株生长发育、叶色、花发育、花色、开花时间、多糖合成、抗病、抗逆和组培快繁特性等基因(表2)，其中，花色、花发育、花期、抗逆和多糖合成的基因数分别为20、19、 15、13和 5个，占比分别为22.0%、20.9%、16.5%、14.3%和5.5%。
表 2 兰花功能基因鉴定Table 2. Identifications of functional genes in orchid性状
Trait基因
Gene来源
Source载体类型
Carrier type受体、结果及文献
Receptor, result and reference花色
Flower
colorDFR 大花蕙兰
‘Rosannagirl Mild’过表达 矮牵牛，转基因植株花色由白色变为粉红色[32] F3′5′H 蝴蝶兰‘TS444’ 瞬时表达和过表达 蝴蝶兰‘TS444’，花色由粉红色变为品红色[33] PeUFGT3 小兰屿蝴蝶兰 RNAi 小兰屿蝴蝶兰，花色出现不同程度褪色[34] PeMYB2/11/12 小兰屿蝴蝶兰 VIGS 小兰屿蝴蝶兰，红色色素、斑点和网纹丧失[35] DseCHS-B 石斛兰‘睡公主’ RNAi 石斛兰‘睡公主’，花青素积累受损[36] CHS 蝴蝶兰‘大辣椒’ RNAi 蝴蝶兰‘大辣椒’，花瓣颜色变淡、色素含量降低[37] MYBx1 蝴蝶兰‘大辣椒’ 过表达 矮牵牛，转基因植株花色素沉积降低[38] OgPSY 文心兰‘柠檬绿’ RNAi 文心兰‘柠檬绿’，叶黄素含量降低，花朵白色[39] DoFLS1 铁皮石斛 过表达 拟南芥，转基因植株花色苷含量降低、黄酮醇含量增
加[40]PeMYB4L 小兰屿蝴蝶兰 VIGS 小兰屿蝴蝶兰，红色素沉积降低[41] DoMYB5，DobHLH24 铁皮石斛 瞬时表达和过表达 烟草和铁皮石斛，花色苷积累增加[42] 开花时间
Flowering
timeOnFT，OnTFL1 文心兰‘南茜’ 过表达 拟南芥，过表达OnFT植株克服晚花缺陷、过表达OnTFL1抑制成花转变[43] PhalCOL 台湾蝴蝶兰 过表达 烟草，转基因植株花期提早[44] CgFT 春兰 过表达 烟草，转基因植株花期提早[45] CsFT 墨兰 过表达 拟南芥，转基因植株花期提前[46] OMADS1 文心兰 过表达 拟南芥和烟草，转基因植株花期提前[47] DoSOC1 铁皮石斛 过表达 拟南芥，转基因植株花期提前[48] PhFT 蝴蝶兰 Fortune Saltzman 过表达 拟南芥，转基因植株克服晚花缺陷[49] PaFT1，PaFD 台湾蝴蝶兰 过表达 拟南芥和水稻，转基因植株开花(抽穗)变早，叶片少 [50] CsFT 墨兰 过表达 拟南芥，转基因植株提前开花[51] DoAP1 石斛兰 Chao Praya Smile 过表达 拟南芥，过表达促进开花 [52] PhPEBPs 蝴蝶兰 Little Gem Stripes 过表达 拟南芥，PhFT1、 PhFT3、 PhFT5和 PhMFT 促进
开花，PhFT6抑制开花[53]花发育
Floral
developmentPeMADS6 小兰屿蝴蝶兰 过表达 拟南芥，转基因植株出现花瓣状萼片，花寿命增加
3~4倍[54]ORAP11/13 蝴蝶兰 Formosa Rose 过表达 烟草，开花提前，叶片和花器官结构改变[55] PeMADS1/7 小兰屿蝴蝶兰 过表达 拟南芥，PeMADS1挽救ag-4突变体花缺陷表型，PeMADS7抑制花被脱落，种子不育增加[56] PhapLFY 台湾蝴蝶兰 过表达 拟南芥，挽救lfy突变体的异常花不育表型[57] CsAG1 墨兰 过表达 拟南芥，转基因植株花瓣同一性被破坏[58] PaSPK1 台湾蝴蝶兰 VIGS 蝴蝶兰，双花序植株比例减少[59] DoAG1/2 石斛兰 Chao Praya Smile 过表达 拟南芥，挽救ag-4突变体花朵缺陷表型[60] PaFYF1/2 台湾蝴蝶兰 过表达和 VIGS 拟南芥和蝴蝶兰，基因过表达延迟拟南芥花衰老和脱落，且对乙烯处理不敏感；基因沉默蝴蝶兰花衰老和脱落提前[61] PeDL1/2 小兰屿蝴蝶兰 VIGS和
瞬时过表达小兰屿蝴蝶兰，基因沉默影响胚珠初始数量；瞬时过表达导致雌蕊胚珠和柱头腔发育异常[62] MIR390a/b/c 春兰 过表达 拟南芥，转基因植株花药长径比增大、柱头分离、花粉量减少、种子败育率增加[63] CemiR396 建兰 过表达 烟草，转基因植株矮小、叶片变窄、花被严重弯曲、花药发育异常、部分不育[64] 续表 2 Continued table 2 性状
Trait基因
Gene来源
Source载体类型
Carrier type受体、结果及文献
Receptor, result and reference多糖合成
Polysaccharide
synthesisDoUGE 铁皮石斛 过表达 拟南芥，转基因植株多糖积累提高、盐胁迫和渗透胁迫耐受性提高[65] DoGMP1 铁皮石斛 过表达 拟南芥，转基因植株甘露糖含量增加、盐胁迫下萌发率提高[66] DoMYB75 铁皮石斛 过表达 拟南芥，转基因种子水溶性多糖含量增加14%[67] DoRWA3 铁皮石斛 过表达 拟南芥，转基因种子、叶片和茎中多糖的乙酰化水平显著提高[68] 抗逆性
Abiotic
stress tolerancePaCDPK1::GUS 蝴蝶兰 ‘TS97K’ 启动子融合GUS 拟南芥，转基因植株PaCDPK1基因表达受寒冷、伤害和病原体攻击诱导[69] CfJMT 蕙兰 过表达 番茄，转基因植株MeJA 生物合成基因转录水平升高[70] DcCIPk24 铁皮石斛 过表达 拟南芥，转基因植株对干旱和盐胁迫耐受性增强[71] DnMSI1 金钗石斛 过表达 拟南芥，转基因植株NaCl 胁迫耐受性降低[72] CgWRKY18 墨兰 过表达 拟南芥，转基因植株根变长，对盐和渗透胁迫耐受性增强[73] PaCBF1 台湾蝴蝶兰 过表达 拟南芥，转基因植株低温胁迫下细胞膜完整性好[74] 抗病性
Disease resistancePaAGO5a/b 台湾蝴蝶兰 VIGS 蝴蝶兰，过表达PaAGO5a/b植株叶片中CymMV和ORSV积累显著降低[75] DcMYC2b 铁皮石斛 过表达 拟南芥，过表达DcMYC2b感染飞燕草菌核病后损伤小[76] 生长发育
Growth and developmentDsCKX1 石斛兰 ‘Sonia’ 过表达 拟南芥，转基因植株生物量降低，根系生长迅速，离体形成根系能力下降[77] cfJMT 蕙兰 过表达 矮牵牛，转基因植株叶片变小，花粉发育异常[78] CgWRKY57 春兰 过表达 拟南芥，转基因植株根长和萌发率降低[79] CgARF1 春兰 过表达 拟南芥和烟草，转基因植株叶片老化，叶绿素含量降低[80] DcWOX4/9/11 铁皮石斛 过表达 拟南芥，DcWOX4过表达导致植株显著矮小，叶缘羽状深裂，花期推迟2周；DcWOX9过表达导致植株矮化，叶缘锯齿状，开花推迟1周，强表型植株雌雄蕊均不育；DcWOX11过表达导致叶缘向下卷曲[81] 叶发育
Leaf developmentDoeREB5 铁皮石斛 过表达 拟南芥，转基因植株叶片形状和叶柄大小改变[82] 叶色
Leaf colorCsERF2 墨兰 过表达 烟草，转基因植株叶绿素含量下降，叶绿体异常[83] 叶片数量
No. of leavesCcMYB24 兰 ‘TRIR’ 过表达 拟南芥，转基植株叶片数量增加[84] 2. 转基因和基因编辑
转基因技术是兰花分子育种核心技术之一。自Kuehnlehe等[104]利用基因枪法将PRV CP基因转入石斛兰原球茎以来，兰花转基因技术研究取得巨大进展(表3)。迄今已建立蝴蝶兰、石斛兰、文心兰、大花蕙兰C. hybrida、春兰、墨兰、寒兰C. kanran、万代兰Vanda和小扇叶兰Erycina pusilla等兰花转基因技术体系，主要转基因方法为农杆菌介导法和基因枪法，涉及的性状包括花型、花色、花期、花寿命、次生代谢物、抗病、抗逆和组培快繁特性等[85-131]。通过过表达外源基因或自身基因、基因沉默、RNAi和激活标签系统等手段已获得花色变深的蝴蝶兰‘TS444’[33]和‘V3’[94]，花期提前的石斛兰[102]，抗除草剂小扇叶兰[89]，多糖和甘露糖含量高、抗逆性强的铁皮石斛[96]，PLB 形成能力增强的蝴蝶兰[97]，顶端分生组织形成多芽和花期提前的石斛兰[98]，花瓣中出现红色斑点的南茜文心兰Oncidium Gower Ramsey[101]和寒兰[103]，鲜切花寿命增加、花蕾脱落延迟的石斛兰[119]，抗病毒的蝴蝶兰[122,124]、石斛兰[104, 123]和抗低温的蝴蝶兰[131]等转基因兰花。
表 3 兰花转基因技术研究状况Table 3. Research status on transgenic technology of orchid方法
Method目标基因
Target gene受体
Receptor载体类型
Carrier type结果及文献
Result and reference农杆菌介导法Agrobacterium-
mediated methodDOH1 石斛 Madame Thong-In自交 F1 过表达 获得154个多芽转化体[85] hptII，gusA 文心兰 Sherry Baby‘OM8’ 过表达 获得28株转基因植株[105] hpt，gfp 蝴蝶兰‘White Hikaru’ 过表达 gfp检出率73.30% [106] GUS 蝴蝶兰 S122-2×S153 和 S153×S119-4的 F1 过表达 获得88株转基因植株[107] ORSV CP 玉花兰 过表达 遗传转化率64.00% [108] nptII，hpt，GUS 兰 ‘RY’、 ‘L4’和‘L23’ 过表达 获得抗潮霉素转基因植株[109] hpt，GUS 石斛兰 Ekapol‘Panda 1’ 过表达 遗传转化率44.40% [110] etr1-1 齿舌兰 Stirling Tiger和
Sweet Sugar过表达 获得转基因植株[86] hpt 大花蕙兰 过表达 遗传转化率62.50% [111] GUS 朵丽蝶兰 Sogo Beach 过表达 获得转基因植株[112] ACS 石斛兰 过表达 遗传转化率12.15% [87] PhaNPR1 台湾蝴蝶兰 基因沉默 获得转基因植株 [88] wheatwin1，wheatwin2，nptII 万代兰 Kasem’s Delight
Tom Boykin过表达 获得转基因植株[113] GUS 墨兰‘企剑白墨’ 过表达 遗传转化率0.75% [114] MSRB7 小扇叶兰 过表达 获得抗除草剂转基因植株，遗传转化率0.60% [89] GUS 石斛兰 Formidible ‘Ugusu’ 过表达 遗传转化率18.50% [115] GUS 金钗石斛 过表达 遗传转化率27.30% [116] eGFP，HptII 台湾蝴蝶兰‘M1663’(4n)、
朵丽蝶兰 Join Angel ‘TH274-1’
(4n)和台湾蝴蝶兰(2n)过表达 遗传转化率1.20%~5.20% [117] C3H，C4H，4CL，CCR，IRX 铁皮石斛 CRISPR/Cas9 遗传转化率93.60% [90] AcF3H 石斛兰‘5N’ 过表达 遗传转化率10.13% [91] KNAT1 毛药石斛 过表达 遗传转化率70.00% [92] DcObgC-GUS/GFP 铁皮石斛 过表达 遗传转化率56.50[93] PeMYB11 蝴蝶兰 Sogo Yukidian‘V3’ 过表达 转基因植株花色素苷积累升高[94] GUS 金钗石斛 过表达 遗传转化率34.44%~82.44% [118] MADS 小兰屿蝴蝶兰 CRISPR/Cas9 获得转基因植株[95] DoUGP 铁皮石斛 过表达 转基因植株总多糖和甘露糖含量升高，抗逆性增强[96] AntiCpACO 石斛兰 Sonia ‘睡公主’ 过表达 转基因植株花蕾脱落延迟，切花寿命增加[119] PaSTM 台湾蝴蝶兰 过表达 转基因植株 PLB再生能力增强[97] 续表 3 Continued table 3 方法
Method目标基因
Target gene受体
Receptor载体类型
Carrier type结果及文献
Result and reference基因枪法
Particle
bombardmentPRV CP 石斛兰‘K1346’、‘K1347’、
‘K1348’和‘K1349’过表达 获得抗病毒植株[104] DOH1 石斛兰 Madame Thong-In 过表达，
反义表达过表达转基因植株芽形成和发育受抑制，反义转基因植株顶端分生组织形成多芽、开花早，遗传转化率60.00%[98] DOMADS1::GUS 石斛兰 Madame Thong-In 基因表达 获得转基因植株[99] GUS 金钗石斛 过表达 愈伤组织和类原球茎转化率分别为12%和2%[120] GFP 石斛兰 Sonia 17 过表达 获得转基因植株[121] CymMV CP 蝴蝶兰 ‘TS340’ 过表达 转基因植株抗Cym MV能力增强[122] CymMV-CS CP 石斛兰 Hickam Deb 过表达 转基因植株抗Cym MV能力增强[123] CymMV CP，Pflp 蝴蝶兰 ‘TS97K’ 过表达 获得双抗转基因植株[124] GUS 石斛兰Jaquelyn Thomas 过表达 遗传转化率19.87% [125] DcOSEP1 石斛兰 Madame Thong-In和
Chao Praya Smile过表达 获得转基因植株[100] GFP 石斛兰 Sonia 17 过表达 遗传转化率1.70% [126] EgTCTP 蝴蝶兰 过表达 转基因植株类原球茎芽分化更慢[127] OgCHS 文心兰‘南茜’ 过表达 转基因植株花瓣出现红色斑点[101] DOFT 石斛兰 Chao Praya Smile 过表达，RNAi 过表达转基因植株开花提早，RNAi植株开花延迟[102] CkCHS-1，CkDFR，CkANS 寒兰 过表达 转基因植株花瓣和唇瓣中出现红色斑点[103] 子房注射法
Ovary
injectionhpt，GUS 金钗石斛 过表达 获得转基因植株[128] GUS, Kan 紫宝石蝴蝶兰 过表达 遗传转化率3.10% [129] 花粉管通道法
Pollen tube
pathwayhptII 蝴蝶兰和朵丽蝶兰 过表达 获得52个转化体[130] AtCBF1 蝴蝶兰 过表达 获得抗低温转基因植株[131] 基因编辑技术是一种有效定向育种技术。Kui等[90]以原球茎为受体，通过农杆菌介导法建立了铁皮石斛CRISPR/Cas9基因编辑技术体系，C3H、C4H、4CL、CCR和IRX基因编辑效率为10%~100%。Tong等[95]以培养30 d的原球茎为受体，对小兰屿蝴蝶兰MADS基因进行编辑，从20个外植体中共获得47个突变体，其中，46个突变体在3个位点均发生突变，总突变率为97.90%。
3. 分子标记辅助选择
高密度遗传连锁图谱构建对QTL定位、基因克隆和分子标记辅助选择等具有重要作用。在石斛兰、蝴蝶兰和兜兰Paphiopedilum等兰花上已构建出了高密度分子遗传图谱(表4)[19, 132-139]。Lu等[135]以细茎石斛D. moniliforme×铁皮石斛杂交后代为作图群体，构建了含 8573个SLAF标记、19个连锁群的石斛兰分子遗传图谱，标记间平均距离为 0.32 cM，鉴定出5个与茎多糖含量相关的QTLs。 Li等[136]以100个金钗石斛和大苞鞘石斛D. wardianum F1杂交后代为作图群体，构建出含9564 个SNP标记、19个连锁群的石斛兰分子遗传图谱，标记间平均距离为 0.41 cM，鉴定出2个与茎长和1个与茎粗相关的QTLs。Hsu等[138]通过测序基因分型(Genotyping-by-sequencing，GBS)鉴定出1191个SNPs，基于1191个SNPs和22个SSRs构建出含19个连锁群的蝴蝶兰高密度分子遗传图谱，通过基因组关联分析(Genome-wide association study，GWAS)，鉴定出7个与1个花部颜色相关联的SNPs。Li等[139]以同色兜兰Paph. concolor和带叶兜兰Paph. hirsutissimum的95个杂种后代为作图群体，构建出具有13个连锁群、8410个SNP标记的遗传图谱，标记间平均距离为0.19 cM，筛选出与叶长、叶宽、叶厚和叶数连锁的SNP标记共12个。
表 4 兰花分子遗传图谱构建Table 4. Construction of genetic map in orchid by molecular marker作图群体(F1)
Mapping
population植株数
No. of
plants标记类型
Type of
marker图谱长度/cM
Length of
genetic map连锁群数
No. of
linkage groups标记间距离/cM
Marker
interval参考文献
Reference铁皮石斛×重唇石斛 Dendrobium
officinale×D. hercoglossum90 RAPD, SRAP ♀629.40,
♂1 304.60♀14,
♂19♀11.20,
♂11.60[132] 铁皮石斛×钩状石斛
D. officinale×D. aduncum140 SRAP, SSR 1 580.40 27 11.89 [133] 金钗石斛×细茎石斛
D. nobile×D. moniliforme90 RAPD, ISSR ♀1 474.00,
♂1 326.50♀15,
♂16♀14.75,
♂14.88[134] 细茎石斛×铁皮石斛
D. moniliforme×D. officinal111 SNP 2 737.49 19 0.32 [135] 金钗石斛×大苞鞘石斛
D. nobile×D. wardianum100 SNP 3612.12 19 0.41 [136] 蝴蝶兰‘462’×蝴蝶兰‘20’
Phalaenopsis ‘462’×P. ‘20’88 AFLP ♀878.30,
♂820.30♀14,
♂15♀5.00,
♂6.70[137] 台湾蝴蝶兰×羞花蝴蝶兰
P. aphrodite×P. modesta184 RAD 3 075.80 22 1.00 [19] 台湾蝴蝶兰×小兰屿蝴蝶兰
P. aphrodite×P. equestris117 SNP l5 192.05 27 0.13 [138] 同色兜兰×带叶兜兰 Paphiopedilum
concolor×Paph. hirsutissimum95 SNP 1 616.18 13 0.19 [139] 王健等[140]利用RAPD分子标记对16种有香和无香春兰进行分析，筛选到引物BA0088，该引物在有香春兰中扩增出一条特异性370 bp条带，带型清晰，重复性好。刘泽强[141]利用大花蕙兰和墨兰杂交后代F1群体筛选到2个与兰花香气性状紧密连锁的ISSR标记b19和b21，b19对兰花有香后代的预测准确率为84%、b21的预测准确率为88%，双标记预测准确率为98%。李晓红[142]筛选到8个与兰花纯黄绿色花连锁的SSR标记，这些标记辅助纯黄绿色花选择的准确率为52%~100%。肖文芳等[143]以黄金豹蝴蝶兰P. Frigdaas Oxford和白天使蝴蝶兰P. Join Angel及其杂交后代为材料，筛选到2个与蝴蝶兰花底色关联的SNP标记，Marker35886和Marker70907的2个SNP位点对花底色的鉴定准确率分别为66.67%和73.33%，双标记鉴定准确率达93.33%。
4. 展望
兰花种类繁多、用途广泛、市场前景广阔。随着经济发展和人们个性化需求的增加，市场对兰花新品种的需求逐渐提高，如何快速高效选育兰花新品种，满足市场需求已成为育种家急需解决的问题。分子育种能够最大限度利用种质资源，快速定向培育兰花新品种，目前已成为兰花育种方法研究的热点[2,5]。随着组学技术的发展，分子育种技术将成为影响兰花新品种选育、决定产业竞争力和高质量发展的关键核心技术。
21世纪以来，兰花分子育种技术研究取得了巨大进步，但鲜见利用分子育种方法培育出兰花新品种的报道，原因是兰花转基因、基因编辑和分子标记辅助选择等技术还处于技术研发阶段，且目前的研究主要是从技术创新而非品种创新的角度进行的。为了将分子育种技术更好地应用于品种创新，今后应加强以下几个方面的研究工作。
1)加强优良商业化品种转基因技术研究，建立稳定高效兰花转基因技术体系。转基因技术不仅是转基因和基因编辑育种的技术基础，也是挖掘兰花主要育种目标性状基因的关键技术。目前兰花转基因技术只在蝴蝶兰、石斛等少数兰花的个别材料中取得成功，而且转基因效率不高，今后应研究其他主要商业化兰花和优良品种的高效转基因技术，为选育具有商业化应用前景的兰花新品种奠定基础。
2)加强遗传图谱构建和标记辅助选择技术研究，构建以SSR、Indel等分子标记为主的高密度分子遗传图谱，建立分子标记辅助主要育种目标性状选择技术体系。虽然目前已构建出几种兰花高密度分子遗传图谱，但这些图谱主要是SNP标记图谱，育种应用价值不高，已建立分子标记辅助选择技术的只有花香和黄绿色花等少数性状，今后应加强筛选与育种早期无法选择或选择难度大、成本高和准确率低的性状紧密连锁分子标记，建立其辅助目标性状选择技术，实现育种早期高效定向选择的目的，以进一步提高育种效率和效益。
3)加强控制主要育种目标性状的主效基因挖掘。不管是转基因还是基因编辑育种，获得功能基因是前提，目前虽然鉴定出许多育种目标性状基因，但这些基因都是通过反向遗传学方法获得的，多数基因的功能未经验证，其效应和遗传规律不清楚，今后应加强采用正向遗传学方法或结合传统遗传学研究结果挖掘关键基因。
4)加强兰花基因编辑技术研究。利用CRISPR/Cas9 编辑兰花基因效率高[90,95]，显示出其在兰花育种上的巨大应用潜力。因此，大力加强兰属兰花、卡特兰、文心兰等兰花基因编辑技术研究、建立基因编辑技术体系，对进一步推动兰花育种和产业高质量发展具有重要意义。
-
表 1 兰花基因组测序
Table 1 Genome sequencing of orchid
物种
Species染色体数
No. of
chromosomes基因组
大小/Gb
Genome size预测基因数
No. of
predicted genes重复序列/%
Repetitive
sequence测序技术及文献
Sequencing technique
and reference墨兰 Cymbidium sinense 20 3.52 29 638 77.78 Illumina HiSeq 2000[7] 建兰 C. ensifolium 20 3.62 29 073 80.58 Illumina HiSeq 2500 PacBio[8] 春兰 C. goeringii 20 3.99 29 556 88.87 Illumina Hiseq 2500[9] 春兰 C. goeringii 20 4.10 29 272 77.65 Illumina HiSeq 2500 PacBio[10] 硬叶兰 C. mannii 20 2.88 27 192 82.80 MGISEQ-2000[11] 铁皮石斛
Dendrobium officinale19 1.35 35 567 63.33 Illumina HiSeq 2000 PacBio[12] 铁皮石斛 D. catenatum 19 1.12 29 257 78.10 Illumina Hiseq2000[13] 鼓槌石斛 D. chrysotoxum 19 1.37 30 044 62.81 MGISEQ-2000 NovaSeqPacBio[14] 铁皮石斛 D. officinale 19 1.23 25 894 76.77 Illumina Hiseq 4000 PacBio[15] 霍山石斛 D. huoshanense 19 1.285 21 070 74.92 Illumina HiSeq 2000 PacBio[16] 金钗石斛 D.nobile 19 1.16 29 476 61.07 Illumina Novaseq6000[17] 小兰屿蝴蝶兰
Phalaenopsis equestris19 1.13 29 545 61.53 Illumina HiSeq 2000[6] 蝴蝶兰‘KHM190’
P. ‘KHM190’19 3.10 41 153 59.74 Illumina HiSeq 2000[18] 台湾蝴蝶兰 P. aphrodite 19 1.03 28 902 60.30 Illumina Hiseq 2000[19] 紫金舌唇兰
Platanthera zijinensis21 4.15 24 513 77.38 Illumina Hiseq 4000 PacBio[20] 广东舌唇兰
Platanthera guangdongensis21 4.27 22 559 82.18 Illumina Hiseq 4000 PacBio[20] 天麻 Gastrodia elata 18 1.06 18 969 66.18 Illumina HiSeq 2500[21] 天麻 G. elata 18 1.12 24 484 68.34 Illumina HiSeq 2000[22] 天麻 G. elata 18 1.04 21 115 66.36 PacBio Sequel II[23] 天麻 G. elata 18 1.05 18 844 74.92 Illumina NovaSeq 6000 PacBio[24] 天麻 G. elata 18 1.09 17 895 63.89 Illumina HiSeq 4000[25] 勐海天麻 G. menghaiensis 18 0.99 17 948 65.08 Illumina HiSeq 2500[26] 白及 Bletilla striata 18 2.37/2.43 26 673 PacBio[27] 香荚兰 Vanilla planifolia 14 1.5 29 167 44.30 Illumina HiSeq 2000[28] 香荚兰 V. planifolia 14 3.4 59 128 72.00 Illumina HiSeq 2000[29] 深圳拟兰
Apostasia shenzhenica34 0.35 21 841 42.05 Illumina HiSeq 2000 PacBio[30] 多枝拟兰 A. ramifera 34 0.37 22 841 44.99 Illumina Hiseq 2000[31] 
下载: 导出CSV
表 2 兰花功能基因鉴定
Table 2 Identifications of functional genes in orchid
性状
Trait基因
Gene来源
Source载体类型
Carrier type受体、结果及文献
Receptor, result and reference花色
Flower
colorDFR 大花蕙兰
‘Rosannagirl Mild’过表达 矮牵牛，转基因植株花色由白色变为粉红色[32] F3′5′H 蝴蝶兰‘TS444’ 瞬时表达和过表达 蝴蝶兰‘TS444’，花色由粉红色变为品红色[33] PeUFGT3 小兰屿蝴蝶兰 RNAi 小兰屿蝴蝶兰，花色出现不同程度褪色[34] PeMYB2/11/12 小兰屿蝴蝶兰 VIGS 小兰屿蝴蝶兰，红色色素、斑点和网纹丧失[35] DseCHS-B 石斛兰‘睡公主’ RNAi 石斛兰‘睡公主’，花青素积累受损[36] CHS 蝴蝶兰‘大辣椒’ RNAi 蝴蝶兰‘大辣椒’，花瓣颜色变淡、色素含量降低[37] MYBx1 蝴蝶兰‘大辣椒’ 过表达 矮牵牛，转基因植株花色素沉积降低[38] OgPSY 文心兰‘柠檬绿’ RNAi 文心兰‘柠檬绿’，叶黄素含量降低，花朵白色[39] DoFLS1 铁皮石斛 过表达 拟南芥，转基因植株花色苷含量降低、黄酮醇含量增
加[40]PeMYB4L 小兰屿蝴蝶兰 VIGS 小兰屿蝴蝶兰，红色素沉积降低[41] DoMYB5，DobHLH24 铁皮石斛 瞬时表达和过表达 烟草和铁皮石斛，花色苷积累增加[42] 开花时间
Flowering
timeOnFT，OnTFL1 文心兰‘南茜’ 过表达 拟南芥，过表达OnFT植株克服晚花缺陷、过表达OnTFL1抑制成花转变[43] PhalCOL 台湾蝴蝶兰 过表达 烟草，转基因植株花期提早[44] CgFT 春兰 过表达 烟草，转基因植株花期提早[45] CsFT 墨兰 过表达 拟南芥，转基因植株花期提前[46] OMADS1 文心兰 过表达 拟南芥和烟草，转基因植株花期提前[47] DoSOC1 铁皮石斛 过表达 拟南芥，转基因植株花期提前[48] PhFT 蝴蝶兰 Fortune Saltzman 过表达 拟南芥，转基因植株克服晚花缺陷[49] PaFT1，PaFD 台湾蝴蝶兰 过表达 拟南芥和水稻，转基因植株开花(抽穗)变早，叶片少 [50] CsFT 墨兰 过表达 拟南芥，转基因植株提前开花[51] DoAP1 石斛兰 Chao Praya Smile 过表达 拟南芥，过表达促进开花 [52] PhPEBPs 蝴蝶兰 Little Gem Stripes 过表达 拟南芥，PhFT1、 PhFT3、 PhFT5和 PhMFT 促进
开花，PhFT6抑制开花[53]花发育
Floral
developmentPeMADS6 小兰屿蝴蝶兰 过表达 拟南芥，转基因植株出现花瓣状萼片，花寿命增加
3~4倍[54]ORAP11/13 蝴蝶兰 Formosa Rose 过表达 烟草，开花提前，叶片和花器官结构改变[55] PeMADS1/7 小兰屿蝴蝶兰 过表达 拟南芥，PeMADS1挽救ag-4突变体花缺陷表型，PeMADS7抑制花被脱落，种子不育增加[56] PhapLFY 台湾蝴蝶兰 过表达 拟南芥，挽救lfy突变体的异常花不育表型[57] CsAG1 墨兰 过表达 拟南芥，转基因植株花瓣同一性被破坏[58] PaSPK1 台湾蝴蝶兰 VIGS 蝴蝶兰，双花序植株比例减少[59] DoAG1/2 石斛兰 Chao Praya Smile 过表达 拟南芥，挽救ag-4突变体花朵缺陷表型[60] PaFYF1/2 台湾蝴蝶兰 过表达和 VIGS 拟南芥和蝴蝶兰，基因过表达延迟拟南芥花衰老和脱落，且对乙烯处理不敏感；基因沉默蝴蝶兰花衰老和脱落提前[61] PeDL1/2 小兰屿蝴蝶兰 VIGS和
瞬时过表达小兰屿蝴蝶兰，基因沉默影响胚珠初始数量；瞬时过表达导致雌蕊胚珠和柱头腔发育异常[62] MIR390a/b/c 春兰 过表达 拟南芥，转基因植株花药长径比增大、柱头分离、花粉量减少、种子败育率增加[63] CemiR396 建兰 过表达 烟草，转基因植株矮小、叶片变窄、花被严重弯曲、花药发育异常、部分不育[64]
下载: 导出CSV
续表 2 Continued table 2 性状
Trait基因
Gene来源
Source载体类型
Carrier type受体、结果及文献
Receptor, result and reference多糖合成
Polysaccharide
synthesisDoUGE 铁皮石斛 过表达 拟南芥，转基因植株多糖积累提高、盐胁迫和渗透胁迫耐受性提高[65] DoGMP1 铁皮石斛 过表达 拟南芥，转基因植株甘露糖含量增加、盐胁迫下萌发率提高[66] DoMYB75 铁皮石斛 过表达 拟南芥，转基因种子水溶性多糖含量增加14%[67] DoRWA3 铁皮石斛 过表达 拟南芥，转基因种子、叶片和茎中多糖的乙酰化水平显著提高[68] 抗逆性
Abiotic
stress tolerancePaCDPK1::GUS 蝴蝶兰 ‘TS97K’ 启动子融合GUS 拟南芥，转基因植株PaCDPK1基因表达受寒冷、伤害和病原体攻击诱导[69] CfJMT 蕙兰 过表达 番茄，转基因植株MeJA 生物合成基因转录水平升高[70] DcCIPk24 铁皮石斛 过表达 拟南芥，转基因植株对干旱和盐胁迫耐受性增强[71] DnMSI1 金钗石斛 过表达 拟南芥，转基因植株NaCl 胁迫耐受性降低[72] CgWRKY18 墨兰 过表达 拟南芥，转基因植株根变长，对盐和渗透胁迫耐受性增强[73] PaCBF1 台湾蝴蝶兰 过表达 拟南芥，转基因植株低温胁迫下细胞膜完整性好[74] 抗病性
Disease resistancePaAGO5a/b 台湾蝴蝶兰 VIGS 蝴蝶兰，过表达PaAGO5a/b植株叶片中CymMV和ORSV积累显著降低[75] DcMYC2b 铁皮石斛 过表达 拟南芥，过表达DcMYC2b感染飞燕草菌核病后损伤小[76] 生长发育
Growth and developmentDsCKX1 石斛兰 ‘Sonia’ 过表达 拟南芥，转基因植株生物量降低，根系生长迅速，离体形成根系能力下降[77] cfJMT 蕙兰 过表达 矮牵牛，转基因植株叶片变小，花粉发育异常[78] CgWRKY57 春兰 过表达 拟南芥，转基因植株根长和萌发率降低[79] CgARF1 春兰 过表达 拟南芥和烟草，转基因植株叶片老化，叶绿素含量降低[80] DcWOX4/9/11 铁皮石斛 过表达 拟南芥，DcWOX4过表达导致植株显著矮小，叶缘羽状深裂，花期推迟2周；DcWOX9过表达导致植株矮化，叶缘锯齿状，开花推迟1周，强表型植株雌雄蕊均不育；DcWOX11过表达导致叶缘向下卷曲[81] 叶发育
Leaf developmentDoeREB5 铁皮石斛 过表达 拟南芥，转基因植株叶片形状和叶柄大小改变[82] 叶色
Leaf colorCsERF2 墨兰 过表达 烟草，转基因植株叶绿素含量下降，叶绿体异常[83] 叶片数量
No. of leavesCcMYB24 兰 ‘TRIR’ 过表达 拟南芥，转基植株叶片数量增加[84]
下载: 导出CSV
表 3 兰花转基因技术研究状况
Table 3 Research status on transgenic technology of orchid
方法
Method目标基因
Target gene受体
Receptor载体类型
Carrier type结果及文献
Result and reference农杆菌介导法Agrobacterium-
mediated methodDOH1 石斛 Madame Thong-In自交 F1 过表达 获得154个多芽转化体[85] hptII，gusA 文心兰 Sherry Baby‘OM8’ 过表达 获得28株转基因植株[105] hpt，gfp 蝴蝶兰‘White Hikaru’ 过表达 gfp检出率73.30% [106] GUS 蝴蝶兰 S122-2×S153 和 S153×S119-4的 F1 过表达 获得88株转基因植株[107] ORSV CP 玉花兰 过表达 遗传转化率64.00% [108] nptII，hpt，GUS 兰 ‘RY’、 ‘L4’和‘L23’ 过表达 获得抗潮霉素转基因植株[109] hpt，GUS 石斛兰 Ekapol‘Panda 1’ 过表达 遗传转化率44.40% [110] etr1-1 齿舌兰 Stirling Tiger和
Sweet Sugar过表达 获得转基因植株[86] hpt 大花蕙兰 过表达 遗传转化率62.50% [111] GUS 朵丽蝶兰 Sogo Beach 过表达 获得转基因植株[112] ACS 石斛兰 过表达 遗传转化率12.15% [87] PhaNPR1 台湾蝴蝶兰 基因沉默 获得转基因植株 [88] wheatwin1，wheatwin2，nptII 万代兰 Kasem’s Delight
Tom Boykin过表达 获得转基因植株[113] GUS 墨兰‘企剑白墨’ 过表达 遗传转化率0.75% [114] MSRB7 小扇叶兰 过表达 获得抗除草剂转基因植株，遗传转化率0.60% [89] GUS 石斛兰 Formidible ‘Ugusu’ 过表达 遗传转化率18.50% [115] GUS 金钗石斛 过表达 遗传转化率27.30% [116] eGFP，HptII 台湾蝴蝶兰‘M1663’(4n)、
朵丽蝶兰 Join Angel ‘TH274-1’
(4n)和台湾蝴蝶兰(2n)过表达 遗传转化率1.20%~5.20% [117] C3H，C4H，4CL，CCR，IRX 铁皮石斛 CRISPR/Cas9 遗传转化率93.60% [90] AcF3H 石斛兰‘5N’ 过表达 遗传转化率10.13% [91] KNAT1 毛药石斛 过表达 遗传转化率70.00% [92] DcObgC-GUS/GFP 铁皮石斛 过表达 遗传转化率56.50[93] PeMYB11 蝴蝶兰 Sogo Yukidian‘V3’ 过表达 转基因植株花色素苷积累升高[94] GUS 金钗石斛 过表达 遗传转化率34.44%~82.44% [118] MADS 小兰屿蝴蝶兰 CRISPR/Cas9 获得转基因植株[95] DoUGP 铁皮石斛 过表达 转基因植株总多糖和甘露糖含量升高，抗逆性增强[96] AntiCpACO 石斛兰 Sonia ‘睡公主’ 过表达 转基因植株花蕾脱落延迟，切花寿命增加[119] PaSTM 台湾蝴蝶兰 过表达 转基因植株 PLB再生能力增强[97]
下载: 导出CSV
续表 3 Continued table 3 方法
Method目标基因
Target gene受体
Receptor载体类型
Carrier type结果及文献
Result and reference基因枪法
Particle
bombardmentPRV CP 石斛兰‘K1346’、‘K1347’、
‘K1348’和‘K1349’过表达 获得抗病毒植株[104] DOH1 石斛兰 Madame Thong-In 过表达，
反义表达过表达转基因植株芽形成和发育受抑制，反义转基因植株顶端分生组织形成多芽、开花早，遗传转化率60.00%[98] DOMADS1::GUS 石斛兰 Madame Thong-In 基因表达 获得转基因植株[99] GUS 金钗石斛 过表达 愈伤组织和类原球茎转化率分别为12%和2%[120] GFP 石斛兰 Sonia 17 过表达 获得转基因植株[121] CymMV CP 蝴蝶兰 ‘TS340’ 过表达 转基因植株抗Cym MV能力增强[122] CymMV-CS CP 石斛兰 Hickam Deb 过表达 转基因植株抗Cym MV能力增强[123] CymMV CP，Pflp 蝴蝶兰 ‘TS97K’ 过表达 获得双抗转基因植株[124] GUS 石斛兰Jaquelyn Thomas 过表达 遗传转化率19.87% [125] DcOSEP1 石斛兰 Madame Thong-In和
Chao Praya Smile过表达 获得转基因植株[100] GFP 石斛兰 Sonia 17 过表达 遗传转化率1.70% [126] EgTCTP 蝴蝶兰 过表达 转基因植株类原球茎芽分化更慢[127] OgCHS 文心兰‘南茜’ 过表达 转基因植株花瓣出现红色斑点[101] DOFT 石斛兰 Chao Praya Smile 过表达，RNAi 过表达转基因植株开花提早，RNAi植株开花延迟[102] CkCHS-1，CkDFR，CkANS 寒兰 过表达 转基因植株花瓣和唇瓣中出现红色斑点[103] 子房注射法
Ovary
injectionhpt，GUS 金钗石斛 过表达 获得转基因植株[128] GUS, Kan 紫宝石蝴蝶兰 过表达 遗传转化率3.10% [129] 花粉管通道法
Pollen tube
pathwayhptII 蝴蝶兰和朵丽蝶兰 过表达 获得52个转化体[130] AtCBF1 蝴蝶兰 过表达 获得抗低温转基因植株[131]
下载: 导出CSV
表 4 兰花分子遗传图谱构建
Table 4 Construction of genetic map in orchid by molecular marker
作图群体(F1)
Mapping
population植株数
No. of
plants标记类型
Type of
marker图谱长度/cM
Length of
genetic map连锁群数
No. of
linkage groups标记间距离/cM
Marker
interval参考文献
Reference铁皮石斛×重唇石斛 Dendrobium
officinale×D. hercoglossum90 RAPD, SRAP ♀629.40,
♂1 304.60♀14,
♂19♀11.20,
♂11.60[132] 铁皮石斛×钩状石斛
D. officinale×D. aduncum140 SRAP, SSR 1 580.40 27 11.89 [133] 金钗石斛×细茎石斛
D. nobile×D. moniliforme90 RAPD, ISSR ♀1 474.00,
♂1 326.50♀15,
♂16♀14.75,
♂14.88[134] 细茎石斛×铁皮石斛
D. moniliforme×D. officinal111 SNP 2 737.49 19 0.32 [135] 金钗石斛×大苞鞘石斛
D. nobile×D. wardianum100 SNP 3612.12 19 0.41 [136] 蝴蝶兰‘462’×蝴蝶兰‘20’
Phalaenopsis ‘462’×P. ‘20’88 AFLP ♀878.30,
♂820.30♀14,
♂15♀5.00,
♂6.70[137] 台湾蝴蝶兰×羞花蝴蝶兰
P. aphrodite×P. modesta184 RAD 3 075.80 22 1.00 [19] 台湾蝴蝶兰×小兰屿蝴蝶兰
P. aphrodite×P. equestris117 SNP l5 192.05 27 0.13 [138] 同色兜兰×带叶兜兰 Paphiopedilum
concolor×Paph. hirsutissimum95 SNP 1 616.18 13 0.19 [139]
下载: 导出CSV
-
[1] 刘仲健, 陈心启, 茹正忠. 中国兰属植物[M]. 北京: 科学出版社, 2006. [2] ZHANG D Y, ZHAO X W, LI Y Y, et al. Advances and prospects of orchid research and industrialization[J]. Horticulture Research, 2022, 9: uhac220. doi: 10.1093/hr/uhac220
[3] HINSLEY A, DE BOER H J, FAY M F. A review of the trade in orchids and its implications for conservation[J]. Botanical Journal of the Linnean Society, 2018, 186(4): 435-455. doi: 10.1093/botlinnean/box083
[4] LI, M M, SU Q L, ZU J R, et al. Triploid cultivars of Cymbidium act as a bridge in the formation of polyploid plants[J]. Frontiers in Plant Science, 2022, 13: 1029915. doi: 10.3389/fpls.2022.1029915
[5] LI C U, DONG N, ZHAO Y M, et al. A review for the breeding of orchids: Current achievements and prospects[J]. Horticultural Plant Journal, 2021, 7(5): 380-392. doi: 10.1016/j.hpj.2021.02.006
[6] CAI J, LIU X, VANNESTE K, et al. The genome sequence of the orchid Phalaenopsis equestris[J]. Nature Genetics, 2015, 47(1): 65-72.
[7] YANG F X, GAO J, WEI Y L, et al. The genome of Cymbidium sinense revealed the evolution of orchid traits[J]. Plant Biotechnology Journal, 2021, 19(12): 2501-2516. doi: 10.1111/pbi.13676
[8] AI Y, LI Z, SUN W H, et al. The Cymbidium genome reveals the evolution of unique morphological traits[J]. Horticulture Research, 2021, 8: 255. doi: 10.1038/s41438-021-00683-z
[9] CHUNG O, KIM J, BOLSER D, et al. A chromosome-scale genome assembly and annotation of the spring orchid (Cymbidium goeringii)[J]. Molecular Ecology Resources, 2022, 22(3): 1168-1177. doi: 10.1111/1755-0998.13537
[10] SUN Y, CHEN G Z, HUANG J, et al. The Cymbidium goeringii genome provides insight into organ development and adaptive evolution in orchids[J]. Ornamental Plant Research, 2021, 1(1): 1-13.
[11] FAN W S, HE Z S, ZHE M Q, et al. High-quality Cymbidium mannii genome and multifaceted regulation of crassulacean acid metabolism in epiphytes[J]. Plant Communications, 2023, 4(5): 100564.
[12] YAN L, WANG X, LIU H, et al. The genome of Dendrobium officinale illuminates the biology of the important traditional Chinese orchid herb[J]. Molecular Plant, 2015, 8(6): 922-934. doi: 10.1016/j.molp.2014.12.011
[13] ZHANG G Q, XU Q, BIAN C, et al. The Dendrobium catenatum Lindl. genome sequence provides insights into polysaccharide synthase, floral development and adaptive evolution[J]. Scientific Reports, 2016, 6: 19029. doi: 10.1038/srep19029
[14] ZHANG Y, ZHANG G Q, ZHANG D, et al. Chromosome-scale assembly of the Dendrobium chrysotoxum genome enhances the understanding of orchid evolution[J]. Horticulture Research, 2021, 8: 183. doi: 10.1038/s41438-021-00621-z
[15] NIU Z, ZHU F, FAN Y, et al. The chromosome-level reference genome assembly for Dendrobium officinale and its utility of functional genomics research and molecular breeding study[J]. Acta Pharmaceutica Sinica B, 2021, 11(7): 2080-2092. doi: 10.1016/j.apsb.2021.01.019
[16] HAN B X, JING Y, DAI J, et al. A chromosome-level genome assembly of Dendrobium huoshanense using long reads and Hi-C data[J]. Genome Biology and Evolution, 2020, 12(12): 2486-2490. doi: 10.1093/gbe/evaa215
[17] XU Q, NIU S C, LI K L, et al. Chromosome-scale assembly of the Dendrobium nobile genome provides insights into the molecular mechanism of the biosynthesis of the medicinal active ingredient of Dendrobium[J]. Frontiers in Genetics, 2022, 13: 844622. doi: 10.3389/fgene.2022.844622
[18] HUANG J Z, LIN C P, CHENG T C, et al. The genome and transcriptome of Phalaenopsis yield insights into floral organ development and flowering regulation[J]. PeerJ, 2016, 4: e2017. doi: 10.7717/peerj.2017
[19] CHAO Y T, CHEN W C, CHEN C Y, et al. Chromosome-level assembly, genetic and physical mapping of Phalaenopsis aphrodite genome provides new insights into species adaptation and resources for orchid breeding[J]. Plant Biotechnology Journal, 2018, 16(12): 2027-2041. doi: 10.1111/pbi.12936
[20] LI M H, LIU K W, LI Z, et al. Genomes of leafy and leafless Platanthera orchids illuminate the evolution of mycoheterotrophy[J]. Nature Plants, 2022, 8(4): 373-388. doi: 10.1038/s41477-022-01127-9
[21] YUAN Y, JIN X H, LIU J, et al. The Gastrodia elata genome provides insights into plant adaptation to heterotrophy[J]. Nature Communications, 2018, 9: 1615. doi: 10.1038/s41467-018-03423-5
[22] CHEN S S, WANG X, WANG Y Z, et al. Improved de novo assembly of the achlorophyllous orchid Gastrodia elata[J]. Frontiers in Genetics, 2020, 11: 580568.
[23] XU Y X, LEI Y T, SU Z X, et al. A chromosome-scale Gastrodia elata genome and large-scale comparative genomic analysis indicate convergent evolution by gene loss in mycoheterotrophic and parasitic plants[J]. The Plant Journal, 2021, 108(6): 1609-1623. doi: 10.1111/tpj.15528
[24] BAE E K, AN C, KANG M J, et al. Chromosome-level genome assembly of the fully mycoheterotrophic orchid Gastrodia elata[J]. G3 Genes Genomes Genetics, 2022, 12(3): jkab433. doi: 10.1093/g3journal/jkab433
[25] WANG Y S, SHAHID M Q. Genome sequencing and resequencing identified three horizontal gene transfers and uncovered the genetic mechanism on the intraspecies adaptive evolution of Gastrodia elata Blume[J]. Frontiers in Plant Science, 2023, 13: 1035157. doi: 10.3389/fpls.2022.1035157
[26] JIANG Y, HU X D, YUAN Y, et al. The Gastrodia menghaiensis (Orchidaceae) genome provides new insights of orchid mycorrhizal interactions[J]. BMC Plant Biology, 2022, 22(1): 179.
[27] JIANG L, LIN M F, WANG H, et al. Haplotype-resolved genome assembly of Bletilla striata (Thunb. ) Reichb. f. to elucidate medicinal value[J]. The Plant Journal, 2022, 111(5): 1340-1353. doi: 10.1111/tpj.15892
[28] HASING T, TANG H B, BRYM M, et al. A phased Vanilla planifolia genome enables genetic improvement of flavour and production[J]. Nature Food, 2020, 1(12): 811-819. doi: 10.1038/s43016-020-00197-2
[29] PIET Q, DROC G, MARANDE W, et al. A chromosome-level haplotype-phased Vanilla planifolia genome highlights the challenge of partial endoreplication for accurate whole genome assembly[J]. Plant Communications, 2022, 3(5): 100330.
[30] ZHANG G Q, LIU K W, LI Z, et al. The Apostasia genome and the evolution of orchids[J]. Nature, 2017, 549: 379-383. doi: 10.1038/nature23897
[31] ZHANG W, ZHANG G, ZENG P, et al. Genome sequence of Apostasia ramifera provides insights into the adaptive evolution in orchids[J]. BMC Genomics, 2021, 22: 536. doi: 10.1186/s12864-021-07852-3
[32] JOHNSON E T, YI H, SHIN B, et al. Cymbidium hybrida dihydroflavonol 4-reductase does not efficiently reduce dihydrokaempferol to produce orange pelargonidin-type anthocyanins[J]. The Plant Journal, 1999, 19(1): 81-85. doi: 10.1046/j.1365-313X.1999.00502.x
[33] SU V, HSU B D. Cloning and expression of a putative cytochrome P450 gene that influences the colour of Phalaenopsis flowers[J]. Biotechnology Letters, 2003, 25(22): 1933-1939. doi: 10.1023/B:BILE.0000003989.19657.53
[34] CHEN W H, HSU C Y, CHENG H Y, et al. Downregulation of putative UDP-glucose: Flavonoid 3-O-glucosyltransferase gene alters flower coloring in Phalaenopsis[J]. Plant Cell Reports, 2011, 30(6): 1007-1017. doi: 10.1007/s00299-011-1006-1
[35] HSU C C, CHEN Y Y, TSAI W C, et al. Three R2R3-MYB transcription factors regulate distinct floral pigmentation patterning in Phalaenopsis spp.[J]. Plant Physiology, 2015, 168: 175-191. doi: 10.1104/pp.114.254599
[36] RATANASUT K, MONMAI C, PILUK P. Transient hairpin RNAi-induced silencing in floral tissues of Dendrobium Sonia ‘Earsakul’ by agroinfiltration for rapid assay of flower colour modification[J]. Plant Cell Tissue and Organ Culture, 2015, 120(2): 643-654. doi: 10.1007/s11240-014-0632-z
[37] 孟妮, 刘雅莉, 窦雪溪, 等. 蝴蝶兰花瓣瞬时转化体系建立[J]. 西北植物学报, 2018, 38(6): 1017-1023. doi: 10.7606/j.issn.1000-4025.2018.06.1017 [38] FU Z, WANG L, SHANG H, et al. An R3-MYB gene of Phalaenopsis, MYBx1, represses anthocyanin accumulation[J]. Plant Growth Regulation, 2019, 88(2): 129-138. doi: 10.1007/s10725-020-00595-3
[39] LIU Y C, YEH C W, CHUNG J D, et al. Petal-specific RNAi-mediated silencing of the phytoene synthase gene reduces xanthophyll levels to generate new Oncidium orchid varieties with white-colour blooms[J]. Plant Biotechnology Journal, 2019, 17(11): 2035-2037. doi: 10.1111/pbi.13179
[40] YU Z, DONG W, DA SILVA J A T, et al. Ectopic expression of DoFLS1 from Dendrobium officinale enhances flavonol accumulation and abiotic stress tolerance in Arabidopsis thaliana[J]. Protoplasma, 2021, 258(4): 803-815. doi: 10.1007/s00709-020-01599-6
[41] WANG R, MAO C J, MING F. PeMYB4L interacts with PeMYC4 to regulate anthocyanin biosynthesis in Phalaenopsis orchid[J]. Plant Science, 2022, 324: 111423. doi: 10.1016/j.plantsci.2022.111423
[42] YANG K, HOU Y B, WU M, et al. DoMYB5 and DobHLH24, transcription factors involved in regulating anthocyanin accumulation in Dendrobium officinale[J]. International Journal of Molecular Sciences, 2023, 24(8): 7552. doi: 10.3390/ijms24087552
[43] HOU C J, YANG C H. Functional analysis of FT and TFL1 orthologs from orchid (Oncidium Gower Ramsey) that regulate the vegetative to reproductive transition[J]. Plant and Cell Physiology, 2009, 50(8): 1544-1557. doi: 10.1093/pcp/pcp099
[44] ZHANG J X, WU K L, TIAN L N, et al. Cloning and characterization of a novel CONSTANS-like gene from Phalaenopsis hybrida[J]. Acta Physiologiae Plantarum, 2011, 33(2): 409-417. doi: 10.1007/s11738-010-0560-4
[45] XIANG L, LI X, QIN D, et al. Functional analysis of FLOWERING LOCUS T orthologs from spring orchid (Cymbidium goeringii Rchb. f.) that regulates the vegetative to reproductive transition[J]. Plant Physiology and Biochemistry, 2012, 58: 98-105. doi: 10.1016/j.plaphy.2012.06.011
[46] HUANG W T, FANG Z M, ZENG S J, et al. Molecular cloning and functional analysis of three FLOWERING LOCUS T (FT) homologous genes from Chinese Cymbidium[J]. International Journal of Molecular Sciences, 2012, 13(9): 11385-11398. doi: 10.3390/ijms130911385
[47] THIRUVENGADAM M, CHUNG I, YANG C. Overexpression of Oncidium MADS box (OMADS1) gene promotes early flowering in transgenic orchid (Oncidium Gower Ramsey)[J]. Acta Physiologiae Plantarum, 2012, 34(4): 1295-1302. doi: 10.1007/s11738-012-0926-x
[48] DING L H, WANG Y W, YU H. Overexpression of DOSOC1, an ortholog of Arabidopsis SOC1, promotes flowering in the orchid Dendrobium Chao Parya Smile[J]. Plant and Cell Physiology, 2013, 54(4): 595-608. doi: 10.1093/pcp/pct026
[49] LI D M, LV F B, ZHU G F, et al. Molecular characterization and functional analysis of a Flowering locus T homolog gene from a Phalaenopsis orchid[J]. Genetics and Molecular Research, 2014, 13(3): 5982-5994. doi: 10.4238/2014.August.7.14
[50] JANG S, CHOI S C, LI H Y, et al. Functional characterization of Phalaenopsis aphrodite flowering genes PaFT1 and PaFD[J]. PLoS One, 2015, 10(8): e0134987. doi: 10.1371/journal.pone.0134987
[51] 黄玮婷, 吴博文, 方中明. 墨兰FT同源基因的时空表达及功能分析[J]. 安徽农业大学学报, 2017, 44(1): 135-141. [52] SAWETTALAKE N, BUNNAG S, WANG Y, et al. DOAP1 promotes flowering in the orchid Dendrobium Chao Praya Smile[J]. Frontiers in Plant Science, 2017, 8: 400.
[53] JIANG L, JIANG X X, LI Y N, et al. FT-like paralogs are repressed by an SVP protein during the floral transition in Phalaenopsis orchid[J]. Plant Cell Reports, 2022, 41(1): 233-248.
[54] TSAI W C, LEE P F, CHEN H I, et al. PeMADS6, a GLOBOSA/PISTILLATA-like gene in Phalaenopsis equestris involved in petaloid formation, and correlated with flower longevity and ovary development[J]. Plant and Cell Physiology, 2005, 46(7): 1125-1139. doi: 10.1093/pcp/pci125
[55] CHEN D, GUO B, HEXIGE S, et al. SQUA-like genes in the orchid Phalaenopsis are expressed in both vegetative and reproductive tissues[J]. Planta, 2007, 226(2): 369-380. doi: 10.1007/s00425-007-0488-0
[56] CHEN Y Y, LEE P F, HSIAO Y Y, et al. C- and D-class MADS-box genes from Phalaenopsis equestris (Orchidaceae) display functions in gynostemium and ovule development[J]. Plant and Cell Physiology, 2012, 53(6): 1053-1067.
[57] JANG S H. Functional characterization of PhapLEAFY, a FLORICAULA/LEAFY ortholog in Phalaenopsis aphrodite[J]. Plant and Cell Physiology, 2015, 56(11): 2234-2247.
[58] SU S H, SHAO X Y, ZHU C F, et al. An AGAMOUS-like factor is associated with the origin of two domesticated varieties in Cymbidium sinense (Orchidaceae)[J]. Horticulture Research, 2018, 5: 48. doi: 10.1038/s41438-018-0052-z
[59] LIN Y J, LI M J, HSING H C, et al. Spike activator 1, encoding a bHLH, mediates axillary bud development and spike initiation in Phalaenopsis aphrodite[J]. International Journal of Molecular Sciences, 2019, 20: 5406. doi: 10.3390/ijms20215406
[60] WANG Y W, LI Y, YAN X J, et al. Characterization of C- and D-class MADS-Box genes in orchids[J]. Plant Physiology, 2020, 184(3): 1469-1481. doi: 10.1104/pp.20.00487
[61] CHEN W H, JIANG Z Y, HSU H F, et al. Silencing of FOREVER YOUNG FLOWER-Like genes from Phalaenopsis orchids promotes flower senescence and abscission[J]. Plant and Cell Physiology, 2021, 62(1): 111-124. doi: 10.1093/pcp/pcaa145
[62] CHEN Y Y, HSIAO Y Y, LI C L, et al. The ancestral duplicated DL/CRC orthologs, PeDL1 and PeDL2, function in orchid reproductive organ innovation[J]. Journal of Experimental Botany, 2021, 72(15): 5442-5461. doi: 10.1093/jxb/erab195
[63] XU Z, LIU Q, CHEN Y, et al. miR390 family of Cymbidium goeringii is involved in the development of reproductive organs in transgenic Arabidopsis[J]. BMC Plant Biology, 2022, 22(1): 149.
[64] YANG F X, LU C Q, WEI Y L, et al. Organ-specific gene expression reveals the role of the Cymbidium ensifolium-miR396/growth-regulating factors module in flower development of the orchid plant Cymbidium ensifolium[J]. Frontiers in Plant Science, 2021, 12: 799778.
[65] YU Z M, HE C M, DA SILVA J A T, et al. Molecular cloning and functional analysis of DoUGE related to water-soluble polysaccharides from Dendrobium officinale with enhanced abiotic stress tolerance[J]. Plant Cell Tissue and Organ Culture, 2017, 131(3): 579-599. doi: 10.1007/s11240-017-1308-2
[66] HE C M, YU Z M, DA SILVA J A T, et al. DoGMP1 from Dendrobium officinale contributes to mannose content of water-soluble polysaccharides and plays a role in salt stress response[J]. Scientific Reports, 2017, 7: 41010. doi: 10.1038/srep41010
[67] HE C M, DA SILVA J A T, WANG H B, et al. Mining MYB transcription factors from the genomes of orchids (Phalaenopsis and Dendrobium) and characterization of an orchid R2R3-MYB gene involved in water-soluble polysaccharide biosynthesis[J]. Scientific Reports, 2019, 9: 13818. doi: 10.1038/s41598-019-49812-8
[68] SI C, DA SILVA J A T, HE C M, et al. DoRWA3 from Dendrobium officinale plays an essential role in acetylation of polysaccharides[J]. International Journal of Molecular Sciences, 2020, 21(17): 6250. doi: 10.3390/ijms21176250
[69] TSAI T M, CHEN Y R, KAO T W, et al. PaCDPK1, a gene encoding calcium-dependent protein kinase from orchid, Phalaenopsis amabilis, is induced by cold, wounding, and pathogen challenge[J]. Plant Cell Reports, 2007, 26(10): 1899-1908. doi: 10.1007/s00299-007-0389-5
[70] XU Q, WANG S, HONG H, et al. Transcriptomic profiling of the flower scent biosynthesis pathway of Cymbidium faberi Rolfe and functional characterization of its jasmonic acid carboxyl methyltransferase gene[J]. BMC Genomics, 2019, 20: 125. doi: 10.1186/s12864-019-5501-z
[71] ZHANG T T, LI Y X, KANG Y Q, et al. The Dendrobium catenatum DcCIPK24 increases drought and salt tolerance of transgenic Arabidopsis[J]. Industrial Crops and Products, 2022, 187: 115375. doi: 10.1016/j.indcrop.2022.115375
[72] CUI B L, HUANG M, GUO C D, et al. Cloning and expression analysis of DnMSI1 gene in orchid species[J]. Plant Signaling and Behavior, 2022, 17(1): e2021649. doi: 10.1080/15592324.2021.2021649
[73] WEI Y L, JIN J P, LIANG D, et al. Genome-wide identification of Cymbidium sinense WRKY gene family and the importance of its group III members in response to abiotic stress[J]. Frontiers in Plant Science, 2022, 13: 969010. doi: 10.3389/fpls.2022.969010
[74] PENG P H, LIN C H, TSAI H W, et al. Cold response in Phalaenopsis aphrodite and characterization of PaCBF1 and PaICE1[J]. Plant and Cell Physiology, 2014, 55(9): 1623-1635. doi: 10.1093/pcp/pcu093
[75] KUO S Y, HU C C, HUANG Y W, et al. Argonaute 5 family proteins play crucial roles in the defence against Cymbidium mosaic virus and Odontoglossum ringspot virus in Phalaenopsis aphrodite subsp. formosana[J]. Molecular Plant Pathology, 2021, 22(6): 627-643. doi: 10.1111/mpp.13049
[76] LI C, CAI X, SHEN Q Y, et al. Genome-wide analysis of basic helix–loop–helix genes in Dendrobium catenatum and functional characterization of DcMYC2 in jasmonate-mediated immunity to Sclerotium delphinii[J]. Frontiers in Plant Science, 2022, 13: 956210. doi: 10.3389/fpls.2022.956210
[77] YANG S, YU H, XU Y, et al. Investigation of cytokinin-deficient phenotypes in Arabidopsis by ectopic expression of orchid DSCKX1[J]. FEBS Letters, 2003, 555(2): 291-296. doi: 10.1016/S0014-5793(03)01259-6
[78] TIAN C, LIU S, JIANG L, et al. The expression characteristics of methyl jasmonate biosynthesis-related genes in Cymbidium faberi and influence of heterologous expression of CfJMT in Petunia hybrida[J]. Plant Physiology and Biochemistry, 2020, 151: 400-410. doi: 10.1016/j.plaphy.2020.03.051
[79] LIU H H, WANG L P, JING X J, et al. Functional analysis of CgWRKY57 from Cymbidium goeringii in ABA response[J]. PeerJ, 2021, 9: e10982. doi: 10.7717/peerj.10982
[80] XU Z H, LI F L, LI M, et al. Functional analysis of ARF1 from Cymbidium goeringii in IAA response during leaf development[J]. PeerJ, 2022, 10: e13077. doi: 10.7717/peerj.13077
[81] 陈凯, 王灏, 陈燚婷, 等. 铁皮石斛WOX家族基因在生长发育中的功能分析[J]. 遗传, 2023, 45(8): 700-714. [82] SUN Y Q, SHEN Y, LI A N, et al. Ectopic expression of Dendrobium EREB5 gene in Arabidopsis influences leaf morphology[J]. In Vitro Cellular and Developmental Biology: Plant, 2014, 50(4): 425-435. doi: 10.1007/s11627-014-9604-6
[83] GAO J, LIANG D, XU Q, et al. Involvement of CsERF2 in leaf variegation of Cymbidium sinense ‘Dharma’[J]. Planta, 2020, 252(2): 29. doi: 10.1007/s00425-020-03426-x
[84] LI G Y, CHENG L J, LI Z L, et al. Over-expression of CcMYB24, encoding a R2R3-MYB transcription factor from a high-leaf-number mutant of Cymbidium, increases the number of leaves in Arabidopsis[J]. PeerJ, 2023, 11: e15490. doi: 10.7717/peerj.15490
[85] YU H, YANG S H, GOH C J. Agrobacterium-mediated transformation of a Dendrobium orchid with the class 1 knox gene DOH1[J]. Plant Cell Reports, 2001, 20: 301-305. doi: 10.1007/s002990100334
[86] RAFFEINER B, SEREK M, WINKELMANN T. Agrobacterium tumefaciens-mediated transformation of Oncidium and Odontoglossum orchid species with the ethylene receptor mutant gene etr1-1[J]. Plant Cell, 2009, 98(2): 125-134.
[87] 冯莹, 赖钟雄. 石斛兰转ACS反义基因抗性原球茎及转化植株的筛选与鉴定[J]. 西北植物学报, 2013, 33(2): 247-253. [88] CHEN J C, LU H C, CHEN C E, et al. The NPR1 ortholog PhaNPR1 is required for the induction of PhaPR1 in Phalaenopsis aphrodite[J]. Botanical Studies, 2013, 54(1): 31. doi: 10.1186/1999-3110-54-31
[89] LEE S H, LI C W, LIAU C H, et al. Establishment of an Agrobacterium-mediated genetic transformation procedure for the experimental model orchid Erycina pusilla[J]. Plant Cell, 2015, 120(1): 211-220.
[90] KUI L, CHEN H T, ZHANG W X, et al. Building a genetic manipulation tool box for orchid biology: Identification of constitutive promoters and application of CRISPR/Cas9 in the orchid, Dendrobium officinale[J]. Frontiers in Plant Science, 2016, 7: 2036.
[91] KHUMKARJORN N, THANONKEO S, YAMADA M, et al. Agrobacterium-mediated transformation of Dendrobium orchid with the flavanone 3-hydroxylase gene[J]. Turkish Journal of Botany, 2017, 41(5): 442-454.
[92] UTAMI E S W, HARIYANTO S, MANUHARA Y S W. Agrobacterium tumefaciens-mediated transformation of Dendrobium lasianthera J. J. Sm: An important medicinal orchid[J]. Journal of Genetic Engineering and Biotechnology, 2018, 16(2): 703-709. doi: 10.1016/j.jgeb.2018.02.002
[93] CHEN J, WANG L, CHEN J B, et al. Agrobacterium tumefaciens-mediated transformation system for the important medicinal plant Dendrobium catenatum Lindl[J]. In Vitro Cellular and Development Biology: Plant, 2018, 54(3): 228-239. doi: 10.1007/s11627-018-9903-4
[94] HSU C C, SU C J, JENG M F, et al. A HORT1 retrotransposon insertion in the PeMYB11 promoter causes harlequin/black flowers in Phalaenopsis orchids[J]. Plant Physiology, 2019, 180(3): 1535-1548. doi: 10.1104/pp.19.00205
[95] TONG C G, WU F H, YUAN Y H, et al. High-efficiency CRISPR/Cas-based editing of Phalaenopsis orchid MADS genes[J]. Plant Biotechnology Journal, 2020, 18(4): 889-891. doi: 10.1111/pbi.13264
[96] CHEN J, WANG L, LIANG H, et al. Overexpression of DoUGP enhanced biomass and stress tolerance by promoting polysaccharide accumulation in Dendrobium officinale[J]. Frontiers in Plant Science, 2020, 11: 533767.
[97] FANG S C, CHEN J C, CHANG P Y, et al. Co-option of the SHOOT MERISTEMLESS network regulates protocorm-like body development in Phalaenopsis aphrodite[J]. Plant Physiology, 2022, 190(1): 127-145. doi: 10.1093/plphys/kiac100
[98] YU H, YANG S H, GOH C J. DOH1, a class 1 knox gene, is required for maintenance of the basic plant architecture and floral transition in orchid[J]. The Plant Cell, 2000, 12(11): 2143-2159. doi: 10.1105/tpc.12.11.2143
[99] YU H, YANG S H, GOH C J. Spatial and temporal expression of the orchid floral homeotic gene DOMADS1 is mediated by its upstream regulatory regions[J]. Plant Molecular Biology, 2002, 49(2): 225-237. doi: 10.1023/A:1014958118852
[100] CHAI D, LEE M S, NG H J, et al. L-methionine sulfoximine as a novel selection agent for genetic transformation of orchids[J]. Journal of Biotechnology, 2007, 131(4): 466-472. doi: 10.1016/j.jbiotec.2007.07.951
[101] LIU X J, CHUANG Y N, CHIOU C Y, et al. Methylation effect on chalcone synthase gene expression determines anthocyanin pigmentation in floral tissues of two Oncidium orchid cultivars[J]. Planta, 2012, 236(2): 449-462.
[102] WANG Y W, LIU L, SONG S Y, et al. DOFT and DOFTIP1 affect reproductive development in the orchid Dendrobium Chao Praya Smile[J]. Journal of Experimental Botany, 2017, 68(21/22): 5759-5722.
[103] ZHOU Z, YING Z, WU Z G, et al. Anthocyanin genes involved in the flower coloration mechanisms of Cymbidium kanran[J]. Frontiers in Plant Science, 2021, 12: 737815. doi: 10.3389/fpls.2021.737815
[104] KUEHNLE A R, SUGII N. Transformation of Dendrobium orchid using particle bombardment of protocorms[J]. Plant Cell Reports, 1992, 11: 484-488.
[105] LIAU C H, YOU S J, PRASAD V, et al. Agrobacterium tumefaciens-mediated transformation of an Oncidium orchid[J]. Plant Cell Reports, 2003, 21(10): 993-998. doi: 10.1007/s00299-003-0614-9
[106] 柴明良, 金斗焕. 农杆菌介导的蝴蝶兰基因转化系统的建立[J]. 园艺学报, 2004, 34(4): 537-539. doi: 10.16420/j.issn.0513-353x.2004.04.035 [107] MISHIBA K I, CHIN D P, MII M. Agrobacterium-mediated transformation of Phalaenopsis by targeting protocorms at an early stage after germination[J]. Plant Cell Reports, 2005, 24(5): 297-303. doi: 10.1007/s00299-005-0938-8
[108] CHEN L, KAWAI H, OKU T, et al. Introduction of Odontoglossum ringspot virus coat protein gene into Cymbidium niveo-marginatum mediated by Agrobacterium tumefaciens to produce transgenic plants[J]. Engei Gakkai Zasshi, 2006, 75(3): 249-255. doi: 10.2503/jjshs.75.249
[109] CHIN D P, MISHIBA K, MII M. Agrobacterium-mediated transformation of protocorm-like bodies in Cymbidium[J]. Plant Cell Reports, 2007, 26(6): 735-743.
[110] 张妙彬, 梁擎中, 肖浩, 等. 农杆菌介导石斛兰遗传转化的研究[J]. 园艺学报, 2008, 35(4): 565-570. [111] 王微, 杨晓伶, 杨婧, 等. 根癌农杆菌介导大花蕙兰遗传转化的研究[J]. 西北植物学报, 2011, 31(1): 27-32. [112] 崔波, 蒋素华, 牛苏燕, 等. 农杆菌介导蝴蝶兰的遗传转化研究[J]. 河南农业大学学报, 2012, 46(6): 642-645. [113] GNASEKARAN P, ANTONY J J J, UDDAIN J, et al. Agrobacterium-mediated transformation of recalcitrant Vanda Kasem’s Delight orchid with higher efficiency[J]. The Scientific World Journal, 2014, 2: 1-10.
[114] 谢利, 王芬, 曾瑞珍, 等. 农杆菌介导的墨兰遗传转化[J]. 生物工程学报, 2015, 31(4): 542-551. [115] PHLAETITA W, CHIN D P, TOKUHARA K, et al. Agrobacterium-mediated transformation of protocorm-like bodies in Dendrobium Formidible ‘Ugusu’[J]. Plant Biotechnology, 2015, 32(3): 225-231.
[116] PHLAETITA W, CHIN D P, OTANG N V, et al. High efficiency Agrobacterium-mediated transformation of Dendrobium orchid using protocorms as a target material[J]. Plant Biotechnology, 2015, 32(4): 323-327. doi: 10.5511/plantbiotechnology.15.0804a
[117] HSING H X, LIN Y J, TONG C G, et al. Efficient and heritable transformation of Phalaenopsis orchids[J]. Botanical Studies, 2016, 57: 30. doi: 10.1186/s40529-016-0146-6
[118] 王昊, 邓柠檬, 张雅文等. 金钗石斛转基因体系的建立[J]. 华南师范大学学报(自然科学版), 2019, 51(2): 62-68. [119] SORNCHAI P, DOORN W G V, IMSABAI W, et al. Dendrobium orchids carrying antisense ACC oxidase: Small changes in flower morphology and a delay of bud abortion, flower senescence, and abscission of flowers[J]. Transgenic Research, 2020, 29(4): 429-442. doi: 10.1007/s11248-020-00209-8
[120] MEN S, MING X, WANG Y, et al. Genetic transformation of two species of orchid by biolistic bombardment[J]. Plant Cell Reports, 2003, 21(6): 592-598. doi: 10.1007/s00299-002-0559-4
[121] TEE C, MARZIAH M, TAN C, et al. Evaluation of different promoters driving the GFP reporter gene and selected target tissues for particle bombardment of Dendrobium Sonia 17[J]. Plant Cell Reports, 2003, 21(5): 452-458.
[122] LIAO L J, PAN I C, CHAN Y L, et al. Transgene silencing in Phalaenopsis expressing the coat protein of Cymbidium mosaic virus is a manifestation of RNA-mediated resistance[J]. Molecular Breeding, 2004, 13(3): 229-242. doi: 10.1023/B:MOLB.0000022527.68551.30
[123] CHANG C, CHEN Y C, HSU Y H, et al. Transgenic resistance to Cymbidium mosaic virus in Dendrobium expressing the viral capsid protein gene[J]. Transgenic Research, 2005, 14(1): 41-46. doi: 10.1007/s11248-004-2373-y
[124] CHAN Y L, LIN K H, SANJAYA, et al. Gene stacking in Phalaenopsis orchid enhances dual tolerance to pathogen attack[J]. Transgenic Research, 2005, 14(3): 279-288.
[125] SUWANAKETCHANATIT C, PILUEK J, PEYACHOKNAGUL S, et al. High efficiency of stable genetic transformation in Dendrobium via microprojectile bombardment[J]. Biologia Plantarum, 2007, 51(4): 720-727. doi: 10.1007/s10535-007-0148-z
[126] TEE S C, MAZIAH M, TAN S C, et al. Selection of co-transformed Dendrobium Sonia 17 using hygromycin and green fluorescent protein[J]. Biologia Plantarum, 2011, 55(3): 572-576. doi: 10.1007/s10535-011-0128-1
[127] KANCHANAPOOM K, NAKKAEW A, KANCHANAPOOM K, et al. Efficient biolistic transformation and regeneration capacity of an EgTCTP transgene in protocorm-like bodies of Phalaenopsis orchid[J]. Notulae Botanicae Horti Agrobotanici Cluj-Napoca, 2012, 40(1): 58-64. doi: 10.15835/nbha4017256
[128] 刘其府, 董会, 曾宋君, 等. 农杆菌子房注射法对金钗石斛的活体转化研究[J]. 华南农业大学学报, 2013, 34(3): 378-382. doi: 10.7671/j.issn.1001-411X.2013.03.019 [129] 张莉, 王佳, 郑枫, 等. 子房注射法转化‘紫宝石’蝴蝶兰的研究[J]. 热带作物学报, 2018, 39(4): 669-674. [130] TSAY H S, HO H M, GUPTA S K, et al. Development of pollen mediated activation tagging system for Phalaenopsis and Doritaenopsis[J]. Electronic Journal of Biotechnology, 2012, 15(4): 9.
[131] WANG R R, YU L, KUANG P, et al. Transfer of the Arabidopsis CBF1, a cold-induced transcription activator, into Phalaenopsis via pollen-tube pathway[J]. Phyton-Annales Rei Botanicae, 2022, 62/63: 87-96.
[132] XUE D W, FENG S G, ZHAO H Y, et al. The linkage maps of Dendrobium species based on RAPD and SRAP markers[J]. Journal of Genetics and Genomics, 2010, 37(3): 197-204. doi: 10.1016/S1673-8527(09)60038-2
[133] LU J J, WANG S, ZHAO H Y, et al. Genetic linkage map of EST-SSR and SRAP markers in the endangered Chinese endemic herb Dendrobium (Orchidaceae)[J]. Genetics and Molecular Research, 2012, 11(4): 4654-4667. doi: 10.4238/2012.December.21.1
[134] FENG S G, ZHAO H Y, LU J J, et al. Preliminary genetic linkage maps of Chinese herb Dendrobium nobile and D. moniliforme[J]. Journal of Genetics, 2013, 92(2): 205-212. doi: 10.1007/s12041-013-0246-y
[135] LU J, LIU Y, XU J, et al. High-density genetic map construction and stem total polysaccharide content-related QTL exploration for Chinese endemic Dendrobium (Orchidaceae)[J]. Frontiers in Plant Science, 2018, 9: 398. doi: 10.3389/fpls.2018.00398
[136] LI J, XU Y, WANG Z. Construction of a high-density genetic map by RNA sequencing and eQTL analysis for stem length and diameter in Dendrobium ( Dendrobium nobile × Dendrobium wardianum )[J]. Industrial Crops and Products, 2019, 128: 48-54. doi: 10.1016/j.indcrop.2018.10.073
[137] XU S P, LIAO F X. A genetic linkage map of Phalaenopsis-based on AFLP markers and the two-way pseudo-testcross mapping strategy[J]. International Journal of Agriculture and Biology, 2017, 19(3): 551-557. doi: 10.17957/IJAB/15.0333
[138] HSU C C, CHEN S Y, CHIU S Y, et al. High-density genetic map and genome-wide association studies of aesthetic traits in Phalaenopsis orchids[J]. Scientific Reports, 2022, 12: 3346. doi: 10.1038/s41598-022-07318-w
[139] LI D M, ZHU G F. High-density genetic linkage map construction and QTLs identification associated with four leaf-related traits in lady’s slipper orchids (Paphiopedilum concolor × Paphiopedilum hirsutissimum)[J]. Horticulturae, 2022, 8(9): 842. doi: 10.3390/horticulturae8090842
[140] 王健, 乐超银, 谢伟, 等. 兰花香味相关基因的RAPD分子标记[J]. 江苏农业科学, 2006, 34(5): 78-79. doi: 10.15889/j.issn.1002-1302.2006.05.030 [141] 刘泽强. 大花蕙兰标记辅助选择育种技术体系的建立[D]. 广州: 华南农业大学, 2009. [142] 李晓红. 兰花花香花色基因鉴定和分子标记辅助花色选择技术研究[D]. 广州: 华南农业大学, 2020. [143] 肖文芳, 李佐, 陈和明, 等. 基于SLAF-BSA技术的蝴蝶兰花底色关联SNP分子标记开发与验证[J]. 中国农业大学学报, 2021, 26(9): 92-100. -
期刊类型引用(1)
1. 张玲玲，房林，李琳，吴坤林，曾晶珏，张佳瑞，康明，曾宋君. 兜兰育种技术研究进展. 广西科学. 2023(06): 1052-1059 . 
百度学术
其他类型引用(1)
计量
- 文章访问数: 665
- HTML全文浏览量: 164
- PDF下载量: 109
- 被引次数: 2
