小麦盐华南象草PpCAD基因的亚细胞定位及其在转基因烟草中的异源表达迫相关基因的克隆与表达分析

该研究根据已克隆的华南象草(Pennisetum purpureum cv.Huanan)肉桂醇脱氢酶(CAD)基因PpCAD的cDNA序列,构建亚细胞定位载体pAN580-PpCAD,用PEG介导法转化象草原生质体,以探究PpCAD蛋白在细胞内的定位;同时构建植物过表达载体pBA002-PpCAD,通过农杆菌介导法在烟草中异源表达,以研究PpCAD基因与植物木质素合成的关系。结果显示:(1)PpCAD定位在象草原生质体的细胞质内;(2)过表达载体pBA002-PpCAD转化烟草后获得27株转基因烟草,其中25株PCR鉴定为阳性;(3)半定量RT-PCR检测6株转基因烟草后发现,PpCAD基因在不同植株的表达量存在差异,通过Southern杂交检测后发现该差异与目的基因插入的拷贝数有关;(4)6株转基因烟草和野生型烟草表型上没有明显差异,除目的基因多拷贝插入的植株OEC6外,木质素含量有不同程度的提高,最高比野生型提高了56.50%。研究表明,PpCAD是一个细胞质蛋白,在烟草中过表达PpCAD能够提高植株木质素含量,表明PpCAD基因参与了植物的木质素合成,可用于象草的木质素调控研究。     

海岛棉GbHCT13基因的功能验证

该研究利用海岛棉‘新海21’和陆地棉ND203以及模式植物拟南芥,通过转基因及荧光定量检测等方法探究海岛棉GbHCT13基因(GenBank 登录号MW048849)在纤维发育中的功能。结果显示：(1)成功构建重组载体pCAMBIA3301 GbHCT13,经农杆菌介导法转化、除草剂抗性基因筛选、荧光定量检测方法鉴定获得转GbHCT13基因拟南芥T₃代植株4株;qRT PCR检测表明,转基因植株中GbHCT13基因表达量较野生型极显著增加。(2)转基因拟南芥过表达GbHCT13基因使植株同一时期的生长较野生型旺盛,株形、叶片数、抽薹数和茎秆表皮毛数量均与野生型存在差异;组织化学分析发现,转GbHCT13基因的拟南芥较野生型茎秆初生木质部生长活跃,导管增粗,次生木质部导管细胞壁横截面积变大,但髓质细胞无明显变化;过表达GbHCT13使拟南芥中木质素合成途径基因发生不同程度改变,其中CAD、CCoAOMT、PAL和4CL与GbHCT13基因的表达呈正相关。(3)经大田筛选、分子鉴定,成功获得转GbHCT13基因棉花植株3株;转GbHCT13基因棉花的棉纤维伸长率增加,纤维强度增大;沉默GbHCT13基因使棉花植株木质素含量降低,茎秆表皮毛数量减少,木质部导管细胞数量减少,导管细胞壁中木质素沉积量降低,而棉株并未发生株高上的明显矮化现象,且木质素合成通路中的CAD、CCoAOMT、CCR、PAL 4个基因的表达均呈降低趋势,说明抑制GbHCT13使得棉花生长代谢受阻,影响纤维发育起始。研究表明,GbHCT13基因能影响棉花植株中木质素合成从而调控纤维的生长发育,其功能与GbHCT13基因在模式植物拟南芥中的基本一致。     

水稻BCAT4基因突变株幼苗对PEG模拟干旱胁迫的响应

以水稻野生型‘日本晴’(NIP)及其BCAT4基因突变体BCAT4 1为材料,在苗期进行PEG 6000模拟干旱处理,分析其对幼苗形态、生长和抗逆生理指标的影响,以探究BCAT4基因在水稻响应干旱胁迫中的作用。结果表明：(1)20% PEG处理后野生型NIP幼苗叶片中BCAT4表达量显著高于对照(处理0 d),复水后幼苗存活率显著高于突变体BCAT4 1。(2)20% PEG处理后,两水稻材料幼苗叶片的相对叶绿素含量下降,脯氨酸和可溶性糖含量上升,抗氧化酶活性先上升后下降,且突变体BCAT4 1中上述各指标均显著低于同期NIP。(3)两材料幼苗叶片中丙二醛和过氧化氢含量及相对电导率随胁迫处理天数增加而上升,且BCAT4 1均显著高于同期NIP。(4)在20% PEG处理后,两水稻材料间根系各形态、生长和生理指标的差异均小于相应叶片。研究发现,BCAT4基因突变加剧了干旱胁迫下水稻幼苗叶片叶绿素含量的下降,抑制了地上部渗透调节物质的积累及抗氧化酶活性上升的幅度,促进了丙二醛和过氧化氢积累以及相对电导率增加,从而降低了水稻的耐旱性。     

拟南芥AtCIPK23基因对烟草抗旱能力的影响

为了探索拟南芥AtCIPK23基因对烟草耐旱能力的影响,对3个转AtCIPK23基因阳性纯合株系KA13、KA14和KA44与野生型烟草K326（对照）进行了自然干旱处理,测定离体叶片的失水速率、叶绿素含量、相对电导率、脯氨酸和可溶性糖含量,并分析了转基因及野生型材料对活性氧的清除能力,对活性氧清除基因NtSOD、NtCAT和NtAPX及干旱胁迫相关基因NtDREB、NtLEA5和NtCDPK2的表达量进行检测。结果表明：（1）转基因烟草离体叶片的失水速率明显低于K326;自然干旱7 d后,野生型K326出现了明显的干旱胁迫症状;干旱7 d进行复水后,转基因株系的复水存活率明显高于K326。（2）转基因株系中的叶绿素、脯氨酸及可溶性糖含量比K326显著提高,电导率则明显降低。（3）野生型烟草K326中H₂O₂的积累量明显高于3个转基因株系,转基因株系中ROS清除机制的3个关键基因NtSOD、NtCAT和NtAPX被诱导上调表达。（4）抗旱相关基因NtDREB、NtLEA5和NtCDPK2仅在转基因烟草中受干旱诱导。研究认为,AtCIPK23基因可能具有提高植物抗旱能力的功能。     

不同基因型转Bt水稻种植对土壤螨类群落的影响

随着转基因技术的飞速发展,越来越多的转基因作物新品种被培育成功并得以推广应用,但转基因作物对非靶标生物的生态安全性问题日益引起人们的广泛关注。为加强转基因作物的生态风险评估,以Cry1Ab纯合基因型转Bt（Bacillus thuringiensis）水稻"克螟稻"和Cry1Ab/Ac融合基因型转Bt水稻"华恢1号"及其对照亲本水稻稻田土壤螨类为研究对象,系统调查研究了纯和基因型和融合基因型转Bt水稻种植下土壤螨类的群落组成、数量动态及其群落多样性的变化。研究结果显示,转Bt水稻对土壤螨类的群落组成无负面影响,仅一些稀有类群（<1%）和常见类群（介于1%和10%）消失或出现。且与对照亲本相比,纯和基因型转Bt水稻中仅上罗甲螨科（Epilohmanniidae）上罗甲螨属（Epilohmannia）的百分比含量显著增加了525%。此外,转Bt水稻与其对照亲本稻田土壤螨类的数量动态、群落多样性、群落均匀度和科属丰富度之间均差异不显著（P > 0.05）。而与纯合基因型转Bt水稻相比,融合基因型转Bt水稻可显著提高大田土壤螨类的数量和科属丰富度（P < 0.05）。可见,融合基因型转Bt水稻种植比纯合基因型转Bt水稻更有利于土壤螨类等非靶标生物的发生及其生物多样性保护。     

OsIMA1增强水稻对镉逆境的适应性

铁(Fe)是植物生长发育所必需的营养元素而镉(Cd)是对植物有害的元素且对植物Fe和Cd的吸收存在拮抗作用。OsIMA是一类正调控水稻Fe吸收的一类小肽,其过表达可以促进Fe的积累。为探究OsIMA是否参与水稻对Cd胁迫的适应性,该研究以水稻为研究材料,利用荧光定量PCR分析了OsIMA基因的表达水平,通过遗传转化和CRISPR/Cas9基因编辑技术构建了OsIMA1过表达植物和ima1突变体植物,评估了OsIMA1过表达和突变体植物在Cd逆境条件下的株高,并利用电联耦合等离子体质谱法测量了根和地上部的Fe和Cd含量。结果表明:(1)Cd处理后,OsIMA1和OsIMA2的转录水平上调。(2)OsIMA1过表达植物比野生型植物对Cd胁迫更耐受。(3)ima1功能缺失突变体比野生型植物对Cd胁迫更敏感。(4)OsIMA1过表达植株根系的Cd含量较高,而ima1突变体植株地上部的Cd含量较高。综上所述,OsIMA1通过限制Cd从根向地上部的转运以增强水稻对Cd逆境的适应能力,该研究结果为定向培育耐Cd作物提供了理论参考。     

棉花肉桂醇脱氢酶基因在棉纤维中的表达及对其结构组分的影响

利用农杆菌介导法获得‘新陆早36号’转棉花肉桂醇脱氢酶基因(GhCAD6)材料,以转基因T₆代植株为试材,对GhCAD6基因在叶片基因组中的整合情况和不同发育阶段棉花纤维中的表达量进行分析,研究该基因对棉纤维中结构多糖和苯丙烷类化合物含量及纤维中苯丙烷类结构单体的影响。结果显示：(1)GhCAD6基因以单拷贝的形式整合到受体棉花基因组中。(2)转基因植株纤维中GhCAD6基因的表达量低于相同发育阶段对照样品,在对照样品中GhCAD6基因的表达量表现为先升高,在20 DPA(开花后天数)时表达量最高,之后下降,而在转基因植株纤维中先上升,并于发育15 DPA时表达量下降,20 DPA时又上升至最高,之后再次下降。(3)成熟纤维中,转GhCAD6基因植株纤维中苯丙烷类化合物含量低于对照,但结构多糖含量的差别不明显。(4)转GhCAD6基因纤维中苯丙烷类结构单体——紫丁香基木质素(S 木质素)和愈疮木基木质素(G 木质素)的比值下降。研究表明,棉花转入GhCAD6基因后,纤维发育(15 DPA)中GhCAD6基因的表达量变化可能导致棉花中苯丙烷类化合物含量及其结构单体比率变化,从而造成棉花纤维品质改变。该研究结果可为深入分析GhCAD6基因在改良棉花纤维品质的作用机理提供理论依据。     

转新型抗虫基因Vip3A棉花新种质的创制

该研究构建植物表达载体pBin438 Vip3A,通过农杆菌介导法转化棉花品种‘冀合713’,将新型抗虫基因Vip3A导入到棉花植株,创制对棉铃虫抗性的转基因棉花新种质。结果表明：(1)PCR检测Vip3A基因已经导入到棉花基因组中且能够稳定遗传。(2)室内抗虫性鉴定表明,与对照相比转基因植株对棉铃虫的抗性显著提高,并获得2株高抗和3株抗棉铃虫的转Vip3A基因株系。(3)Southern blotting结果显示,转基因株系BV01为单拷贝。(4)Elisa检测表明,外源Vip3A基因在BV01的根、茎、叶、花、种子中都有表达,在叶片中的Vip3A蛋白表达为苗期>蕾期>花期>铃期>吐絮期。该研究创制了新型抗虫转基因棉花材料,为培育棉花新型抗虫品种提供了种质资源。     

拟南芥agl16突变体的创制及功能鉴定

AGL16是调控拟南芥气孔密度和ABA含量的重要负调控转录因子,在拟南芥抗旱反应过程中发挥重要的作用。为了获取拟南芥agl16突变体材料,采用棉花叶皱缩病毒(CLCrV)介导的VIGE系统筛选靶向敲除拟南芥AGL16的sgRNA,同时利用农杆菌介导的浸花法将完整编辑载体转化野生型拟南芥,创制了拟南芥agl16突变体并进行抗旱性鉴定。结果表明：(1)利用CLCrV介导的VIGE系统筛选获得2个能靶向敲除AGL16基因的sgRNA,同时构建AtU6 26∷AtAGL16 sgRNA1 35S∷Cas9 Ter p1300编辑载体转化野生型拟南芥Col 0,筛选获得靶位点缺失4 bp的agl16纯合突变体(AGL16: 4)。(2)抗旱表型性鉴定结果显示,干旱处理18 d后,大部分野生型植株干枯死亡,而突变体植株受胁迫的表型相对较轻;复水后,野生型和突变体植株的存活率分别为34.5%(10/29)和75%(27/36)。(3)与野生型相比,干旱胁迫下AGL16: 4纯合突变体植株叶片单位面积气孔数量减少、离体叶片失水率显著降低,但二者的单株种子量并没有发生明显的改变。研究认为,AGL16: 4纯合突变体的抗旱性较野生型明显增强,且种子量与野生型基本一致,表明AGL16基因可作为作物抗旱育种理想的候选靶基因;所获得的agl16突变体为后期从农作物中克隆的AGL16同源基因进行功能回补验证提供了有利的转基因受体材料。     

RNAi介导珠子参PjCAS基因沉默对皂苷合成的影响

该研究利用Gateway技术构建珠子参环阿屯醇合成酶基因(Panax japonicus cycloartenol synthase,PjCAS)的RNAi表达载体,利用农杆菌介导转化在珠子参细胞中成功实现了PjCAS 的RNA干扰;采用实时荧光定量PCR分析珠子参皂苷生物合成途径中关键酶基因的表达情况,同时检测转基因细胞中皂苷和植物甾醇含量的变化,探讨PjCAS基因对珠子参皂苷合成的调控作用。结果表明：(1)成功获得PjCAS基因的RNAi片段,并成功构建了PjCAS基因RNAi载体pHellsgate PjCAS。(2)经农杆菌遗传转化,获得6株实现PjCAS基因RNA干扰的转基因阳性细胞系。(3)与普通细胞系相比,转基因细胞系中PjCAS基因的表达量大约下降了85%,同时与珠子参皂苷合成直接相关的关键酶基因PjDS、PjAS表达量最高分别上调了90%和150%。(4)转基因细胞系中6种单体皂苷的含量均显著高于对照组,其中达玛烷型单体皂苷Re、Rb1、Rd和齐墩果烷型单体皂苷R0、IV、IVa的平均含量比普通珠子参细胞系分别提高了28%、49%、40%、36%、59%、50%。说明珠子参皂苷含量的变化受PjCAS基因的间接调控。(5)6株转基因细胞系中植物甾醇含量较对照显著降低了53%~73%。研究发现,沉默PjCAS基因可促进珠子参皂苷合成的关键酶基因PjDS、PjAS显著上调表达,并提高转PjCAS基因细胞系中单体皂苷的含量,从而促进了珠子参皂苷合成量的显著增加,证明通过抑制植物甾醇合成通路关键基因PjCAS的表达可以有效降低植物甾醇合成支路的代谢通量,使更多的代谢流朝着珠子参皂苷合成方向流动,最终促进了珠子参皂苷的生物合成。     

Foreign exploration for Scirtothrips perseae Nakahara (Thysanoptera: Thripidae) and associated natural enemies on avocado (Persea americana Miller)

Scirtothrips perseae Nakahara was discovered attacking avocados in California, USA, in 1996. Host plant surveys in California indicated that S. perseae has a highly restricted host range with larvae being found only on avocados, while adults were collected from 11 different plant species. As part of a management program for this pest, a “classical” biological control program was initiated and foreign exploration was conducted to delineate the home range of S. perseae, to survey for associated natural enemies and inventory other species of phytophagous thrips on avocados grown in Mexico, Guatemala, Costa Rica, the Dominican Republic, Trinidad, and Brazil. Foreign exploration efforts indicate that S. perseae occurs on avocados grown at high altitudes (>1500 m) from Uruapan in Mexico south to areas around Guatemala City in Guatemala. In Costa Rica, S. perseae is replaced by an undescribed congener as the dominant phytophagous thrips on avocados grown at high altitudes (>1300 m). No species of Scirtothrips were found on avocados in the Dominican Republic, Trinidad, or Brazil. In total, 2136 phytophagous thrips were collected and identified, representing over 47 identified species from at least 19 genera. The significance of these species records is discussed. Of collected material 4% were potential thrips biological control agents. Natural enemies were dominated by six genera of predatory thrips (Aeolothrips, Aleurodothrips, Franklinothrips, Leptothrips, Scolothrips, and Karnyothrips). One genus each of parasitoid (Ceranisus) and predatory mite (Balaustium) were found. Based on the results of our sampling techniques, prospects for the importation of thrips natural enemies for use in a “classical” biological control program in California against S. perseae are not promising.     

Structural Peculiarities of Na+,K+-ATPase Isozymes from the Calf Brain

Functionally active preparations of Na⁺,K⁺-ATPase isozymes from calf brain that contain catalytic subunits of three types (1, 2, and 3) were obtained using two approaches: a selective removal of contaminating proteins by the Jorgensen method and a selective solubilization of the enzyme with subsequent reconstitution of their membrane structure by the Esmann method. The ouabain inhibition constants were determined for the isozymes. The real isozyme composition of the Na⁺ pump from the grey matter containing glial cells and the brain stem containing neurons was determined. The plasma membranes of glial cells were shown to contain mainly Na⁺,K⁺-ATPase of the 11 type and minor amounts of isozymes of the 22(1) and the 31(2) type. The axolemma contains 21 and 31 isozymes. A carbohydrate analysis indicated that 11 enzyme preparations from the brain grey matter substantially differ from the renal enzymes of the same composition in the glycosylation of the 1 isoform. An enhanced sensitivity of the 3 catalytic subunit of Na⁺,K⁺-ATPase from neurons to endogenous proteolysis was found. A point of specific proteolysis in the amino acid sequence PNDNR⁴⁹² Y⁴⁹³ was localized (residue numbering is that of the human 3 subunit). This sequence corresponds to one of the regions of the greatest variability in 1-, 2-, 3-, and 4-subunits, but at the same time, it is characteristic of the 3 isoforms of various species. The presence of the 3 isoform of tubulin (cytoskeletal protein) was found for the first time in the high-molecular-mass Na⁺,K⁺-ATPase 31 isozyme complex isolated from the axolemma of brain stem neurons, and its binding to the 3 catalytic subunit was shown.     

Characterization of SSEA-1 glycolipids from the brain of a patient with fucosidosis

Neutral glycolipids from the brain of a patient with Fucosidosis were analyzed and two complex glycolipids containing five and eight sugars were isolated from the cortical grey matter. These two glycolipids reacted with antibodies recognizing the SSEA-1 [Le^x(X)] carbohydrate determinant. SSEA-1 glycolipids are normally expressed in human embryonic brain but are found in only small amounts in postnatal human brain. The accumulation of the two SSEA-1 glycolipids in Fucosidosis brain thus represents a defect which affects the normal developmentally regulated decrease in postnatal, expression of these glycolipids, and may be a contributing factor in the abnormal brain development associated with the disease. Chemical characterization of the two isolated glycolipids by gas chromatographic and mass spectrometric analyses has identified the two glycolipids as lacto-N-fucopentaosylceramide (III) and difucosyl-neolactonorhexaosylceramide.Abbreviations DCl direct chemical ionization - FAB tastatiom bombardment - GC gas chromatography - GSLs glycosphingolipids - MS mass spectrometry - SSEA-1 stage specific embryonic antigen-1 - TLC thin layer chromatographys     

1H and13C-NMR analysis of four pentasaccharides from urine of blood-group A and B,Leb adults. Confirmation of the structure of two new oligosaccharides: Fuc(α1–2)[GalNAc(α1–3)]Gal(β1–3)[Fuc(α1–4)]Glc and Fuc(α1–2)[Gal(α1–3)]Gal(β1–3)[Fuc(α1–4)]Glc

The major pentasaccharides Fuc(1-2)[GalNAc(1-3)]Gal(1-4)[Fuc(1-3)]Glc and Fuc(1-2) [Gal(1-3)]Gal(1-4)[Fuc(1-3)]Glc, which are normally present in the urine of bloodgroup A Le^b and B Le^b healthy subjects, were each found to be contaminated by a minor component when analysed by¹H-NMR. The determination of these structures, Fuc(1-2) [GalNAc(1-3)]Gal(1-3)[Fuc(1-4)]Glc and Fuc(1-2) [Gal(1-3)]Gal(1-3)[Fuc(1-4)]Glc, was based on the results of methylation analysis and¹H/¹³C-NMR spectroscopy.Abbreviations HPLC high performance liquid chromatography - GLC gas liquid chromatography - NMR nuclear magnetic resonance - COSY correlation spectroscopy - Gal d-galactopyranose - GalNAc 2-acetamido-2-deoxy-d-galactopyranose - Glc d-glucopyranose - Fuc l-fucopyranose - LNDFH I lacto-N-difucohexaose I (Le^b determinant     

The evolution of the Hox cluster: insights from outgroups

Two burgeoning research trends are helping to reconstruct the evolution of the Hox cluster with greater detail and clarity. First, Hox genes are being studied in a broader phylogenetic sampling of taxa: the past year has witnessed important new data from teleost fishes, onychophorans, myriapods, polychaetes, glossiphoniid leeches, ribbon worms, and sea anemones. Second, commonly accepted notions of animal relationships are being challenged by alternative phylogenetic hypotheses that are causing us to rethink the evolutionary relationships of important metazoan lineages, especially arthropods, annelids, nematodes, and platyhelminthes.     

N-andO-glycosylation of glycoprotein C synthesized by Herpes simplex virus type 1-infected ricin resistant cells

The progeny of Herpes simplex virus type 1 (HSV-1) grown in ricin-resistant 14 cells (Ric^R14) lackingN-acetylglucosaminyltransferase I was released in the extracellular medium at a very low rate. By using a monoclonal antibody immobilized on Sepharose we purified from HSV-1-infected Ric^R14 cells a viral glycoprotein (gC), which carries bothN-andO-linked oligosaccharides. Glycopeptides obtained from [³H]mannoselabeled gC by Pronase digestion were entirely susceptible to endo--N-acetylglucosaminidase H, and the major oligosaccharide released was Man₄GlcNAc. The accumulation of this high-mannose species was related to the enzymic defect of the host cells and to the long retention of the viral glycoprotein within the cells. The extent ofO-glycosylation evaluated in [¹⁴C]glucosamine-labeled gC from Ric^R14 cells as compared to that of gC from wild type cells did not appear to be significantly modified.Abbreviations Con A concanavalin A - BHK cells baby hamster kidney cells - HSV Herpes simplex virus     

Elucidation of Structure of Lipid A from the Marine Gram-Negative Bacterium Pseudoalteromonas haloplanktis ATCC 14393T

The chemical structure of lipid A, from the marine -proteobacterium Pseudoalteromonas haloplanktis 14393, a main product of lipopolysaccharide hydrolysis (1% AcOH), was determined using chemical methods and NMR spectroscopy. The lipid A was shown to be -1,6-glucosaminobiose 1,4-diphosphate acylated with two (R)-3-hydroxyalkanoic acid residues at C3 and C3 and amidated with one (R)-3-hydroxydodecanoyl and one (R)-3-dodecanoyloxydodecanoyl residue at N2 and N2, respectively.     

Phylogeny of cardinalfishes (Teleostei: Gobiiformes: Apogonidae) and the evolution of visceral bioluminescence

The cardinalfishes (Apogonidae) are a diverse clade of small, mostly reef-dwelling fishes, for which a variety of morphological data have not yielded a consistent phylogeny. We use DNA sequence to hypothesize phylogenetic relationships within Apogonidae and among apogonids and other acanthomorph families, to examine patterns of evolution including the distribution of a visceral bioluminescence system. In conformance with previous studies, Apogonidae is placed in a clade with Pempheridae, Kurtidae, Leiognathidae, and Gobioidei. The apogonid genus Pseudamia is recovered outside the remainder of the family, not as sister to the superficially similar genus Gymnapogon. Species sampled from the Caribbean and Western Atlantic (Phaeoptyx, Astrapogon, and some Apogon species) form a clade, as do the larger-bodied Glossamia and Cheilodipterus. Incidence of visceral bioluminescence is found scattered throughout the phylogeny, independently for each group in which it is present. Examination of the fine structure of the visceral bioluminescence system through histology shows that light organs exhibit a range of morphologies, with some composed of complex masses of tubules (Siphamia, Pempheris, Parapriacanthus) and others lacking tubules but containing chambers formed by folds of the visceral epithelium (Acropoma, Archamia, Jaydia, and Rhabdamia). Light organs in Siphamia, Acropoma, Pempheris and Parapriacanthus are distinct from but connected to the gut; those in Archamia, Jaydia, and Rhabdamia are simply portions of the intestinal tract, and are little differentiated from the surrounding tissues. The presence or absence of symbiotic luminescent bacteria does not correlate with light organ structure; the tubular light organs of Siphamia and chambered tubes of Acropoma house bacteria, those in Pempheridae and the other Apogonidae do not.     

Molecular phylogeny of Phebalium (Rutaceae: Boronieae) and related genera based on the nrDNA regions ITS 1+2

Parsimony analyses of the internal transcribed spacer regions of nuclear ribosomal DNA (ITS 1 & ITS 2) for 38 taxa sampled from the Phebalium group (Rutaceae: Boronieae) and two outgroups confirm that, with the exception of Phebalium sensu stricto and Rhadinothamnus, six of the currently recognised genera within the group are monophyletic. The data indicate that Phebaliums. str. is paraphyletic with respect to Microcybe, and Rhadinothamnus is paraphyletic with respect to Chorilaena. Rhadinothamnus and Chorilaena together are the sister group to Nematolepis. Drummondita, included as an outgroup taxon, clustered within the ingroup as sister to Muiriantha and related to Asterolasia.The phylogeny suggests that the evolution of major clades within a number of these genera (e.g. Phebalium) relates to vicariance events between eastern and south-western Australia. Leionema is an eastern genus, with the most basal taxon being the morphologically distinct Leionema ellipticum from northern Queensland. Leionema also includes one species from New Zealand, but this species (as with some others) proved difficult to sequence and its phylogenetic position remains unknown. Taxonomic changes at the generic level are recommended.The authors wish to thank Paul G.Wilson, PERTH, for advice and discussion, and Paul Forster, BRI, for collecting and providing material of Leionema ellipticum. The project was supported by a Melbourne University Postgraduate Award (to BM), the Australian Biological Resources Study (ABRS), Australian Systematic Botany Society and Wolf Den (Australia) Investments.