皂荚EST-SSR分子标记开发与分析评价

本研究旨在开发皂荚EST-SSR分子标记,为今后皂荚种质资源评价与分析提供基础。首先通过对已公布的皂荚转录组数据进行拼接得到41003个Unigenes,总长度70.4 Mb,平均长度1716 bp,N50为2533 bp。进一步在7009个Unigenes中检测到8494个EST-SSR位点,其中1200条Unigenes含有2个及以上的SSR位点,复合型SSRs有369个。针对所有EST-SSR位点设计得到6494条特异性引物,随机挑选60个位点进行试验验证与分析,其中44对引物可扩增出特异性片段,17对具有多态性,PIC值范围为0.195~0.742,均值为0.501,且大部分多态性位点位于UTR区域。通过对皂荚近缘种进行跨种PCR扩增试验,结果显示在开发的44对有效引物中,9个近缘种美国皂荚、日本皂荚、绒毛皂荚、滇皂荚、华南皂荚、小果皂荚、野皂荚、肥皂荚和美国肥皂荚分别有32、31、23、24、7、40、25、18和18对引物可获得有效扩增片段,说明EST-SSR标记在皂荚近缘种之间具有良好的通用性。研究表明利用转录组数据挖掘的EST-SSR位点具有扩增稳定、多态性良好、近缘种间通用等优点,是林木物种开发分子标记的有效途径之一。     

中国板栗EST-SSR分子标记在栲树中的通用性分析

简单重复序列也称为微卫星分子标记,不仅在同属近缘种间具有良好的通用性,甚至在近缘属间也具有一定的通用性。本研究利用壳斗科基因组信息数据库中公布的中国板栗124对多态的EST-SSR引物在栲树中进行跨属(栗属到栲属)通用性研究,结果显示中国板栗EST-SSR引物在栲树中通用性和多态性分别为42.7%和56.6%;使用19对多态的EST-SSR引物对4个栲树自然居群的遗传多样性进行初步分析,结果显示栲树自然居群具有较高的遗传多样性(Na=6.105,Ho=0.563,He=0.621)。这些引物为栲树群体遗传学的深入研究提供了有力工具。     

Transferability analysis of EST-SSR markers of Castanea mollissima to Castanopsis fargesii

简单重复序列也称为微卫星分子标记,不仅在同属近缘种间具有良好的通用性,甚至在近缘属间也具有一定的通用性.本研究利用壳斗科基因组信息数据库中公布的中国板栗124对多态的EST-SSR引物在栲树中进行跨属(栗属到栲属)通用性研究,结果显示中国板栗EST-SSR引物在栲树中通用性和多态性分别为42.7％和56.6％;使用19对多态的EST-SSR引物对4个栲树自然居群的遗传多样性进行初步分析,结果显示栲树自然居群具有较高的遗传多样性(Na=6.105,Ho=0.563,He=0.621).这些引物为栲树群体遗传学的深入研究提供了有力工具.     

白菜EST-SSR标记的通用性

EST-SSR是从表达序列标签(expressedsequencetag,EST)中开发的新型简单序列重复(simplesequencerepeat,SSR)标记。根据白菜EST设计了15对SSR引物,对白菜、油菜、玉米、高粱、水稻和茶树等进行了PCR,研究了白菜的EST-SSR标记在不同物种间的通用性。所设计的引物对不同白菜品种、近缘种油菜和远缘种玉米、高粱、水稻和茶树的扩增成功率分别为100%、93.3%、80%、93.3%、93.3%和86.7%。在15对引物中,有11对在远缘种中都有扩增产物,而且一些引物可显示多态性,多态性引物分别占了可扩增引物的33.3%、28.6%、28.6%和61.5%。这些结果表明,白菜EST-SSR引物具有较高的通用性,这对于比较基因组学研究有重要意义。     

利用苹果EST-SSR分析木瓜属种质遗传多样性北大核心CSCD

利用苹果属的EST-SSR标记对33份木瓜种质资源进行遗传多样性研究,以分析引物在木瓜属中的通用性。筛选出15对引物进行PCR扩增,其中9对引物显示多态性,共扩增出71条带,其中多态性条带59条,多态性条带比率83.10%,并且可成功区分不同种。PIC多态性信息含量平均值为0.45,Nei’s基因多样性(H)、Shannon指数(I)的平均值分别为0.45、0.71。根据EST-SSR数据的UPGMA聚类分析将材料分为五大类,木瓜属的不同基原样本各聚为一支,能很好地将5个种植物区分,显示出单系性。研究结果表明,苹果属的EST-SSR标记在木瓜属上具有高度的可转移性,可应用于木瓜属植物的资源评价及遗传关系研究。     

利用苹果EST-SSR分析木瓜属种质遗传多样性

利用苹果属的EST-SSR标记对33份木瓜种质资源进行遗传多样性研究,以分析引物在木瓜属中的通用性。筛选出15对引物进行PCR扩增,其中9对引物显示多态性,共扩增出71条带,其中多态性条带59条,多态性条带比率83.10%,并且可成功区分不同种。PIC多态性信息含量平均值为0.45,Nei’s基因多样性(H)、Shannon指数(I)的平均值分别为0.45、0.71。根据EST-SSR数据的UPGMA聚类分析将材料分为五大类,木瓜属的不同基原样本各聚为一支,能很好地将5个种植物区分,显示出单系性。研究结果表明,苹果属的EST-SSR标记在木瓜属上具有高度的可转移性,可应用于木瓜属植物的资源评价及遗传关系研究。     

基于苹果EST-SSR的梨种质资源遗传多样性分析

利用来自苹果的8对EST-SSR标记对48份梨(Pyrus)种质资源进行遗传多样性研究,以分析其在梨属植物上的通用性.结果表明,8对EST-SSR引物在供试材料上均能扩增出与苹果大小相似的产物,所有引物共检测到140个基因位点,其中多态性位点129个,多态性比例为92.14%,并且可成功区分不同品种.根据EST-SSR标记所揭示的多态性和UP-GMA法聚类分析,48份梨种质资源在相似系数0.62处可分为东方梨和西方梨两个种群,而中国的白梨(Pyrus bretschneideri Rehd.)、砂梨(P.pyrifolia Burm.f.Nakai)和秋子梨(P.ussuriensis Maxim.)相互交错在一起,没有独自成组.可见,苹果的EST-SSR标记在梨上具有高度的可转移性,可应用于梨属植物的资源评价及遗传关系研究.     

杂交兰转录组SSR信息分析及EST-SSR标记开发应用

该研究主要开发筛选适用于杂交兰的EST-SSR引物,为杂交兰种质资源评价和遗传变异研究等提供可靠的分子标记。该研究对杂交兰进行转录组高通量测序,挖掘SSR位点和开发EST-SSR标记,并对不同种质的遗传多样性进行分析。结果表明,从31724条杂交兰Unigene中检测出18603个SSR位点,SSR出现频率为58.64%;SSR位点中的主导类型是单核苷酸重复,占总SSR的65.10%,其次是二核苷酸(23.56%)和三核苷酸(10.76%)重复;优势重复基元为A/T、AG/CT、AT/AT和AAG/CTT,分别占总位点的64.72%、13.74%、8.19%和2.51%。利用Primer Premier 5.0共设计了565对SSR引物,从筛选出的64对有效扩增引物中随机选择28对引物,对40份杂交兰种质进行多态性验证与遗传关系分析,其中16对(占57.14%)引物表现出可重复的高多态性,平均多态信息量(PIC)达0.789。基于扩增的多态性SSR信息,40份种质资源可聚为4类,聚类结果与其遗传背景基本一致。该研究印证了转录组测序获得的Unigene是SSR标记开发的有效来源,开发的EST-SSR引物可为杂交兰及近缘种的良种鉴别、遗传图谱构建、分子标记辅助育种及功能基因挖掘等提供有价值的候选标记。     

濒危植物华木莲核基因组微卫星引物开发研究

华木莲（ Sinomanglietia glauca）是在江西宜春和湖南永顺发现的一种珍稀植物。为进一步探讨华木莲群体遗传学特征,本研究采用FIASCO法（fast isolation by AFLP sequences containing repeats）开发了七对华木莲微卫星引物,选取华木莲4个野生居群16个样品对每个位点进行多态性初步检测。结果发现,七个微卫星位点的等位基因数目（NA）为2～7个,观测杂合度（H0）0．079～0．989,期望杂合度（HE）0．187～0．600。另外,使用九个华木莲近缘种对七对引物进行通用性检测,发现这些引物在醉香含笑中通用性最低（28．5％）,在观光木中通用性最高（85．7％）。本文开发的多态性微卫星标记将用于华木莲的繁育系统、群体遗传学等研究,同时也将用于木兰科其它近缘种的群体遗传学研究。     

燕麦EST-SSR标记的开发和利用

为拓展分子标记在燕麦种质资源分析与鉴定中的应用,利用公共数据库中的25376条EST(expressed sequence tags)序列,开展了燕麦EST-SSR功能性标记的开发和利用研究。25376条EST序列经拼接去冗余后获得了11618条序列,从中筛选出含有不同重复基元的SSR且重复次数较多、长度较长的556条EST序列进行引物设计,开发了50对燕麦EST-SSR引物,通过筛选得到40对有效的EST-SSR引物。选取其中4对引物对5个燕麦种质资源进行了PCR扩增及产物测序,结果表明扩增条带多态性是由SSR差异造成的。利用40对ESTSSR引物对15个六倍体燕麦种质资源进行遗传多样性分析,共扩增出89个等位基因,平均每对引物产生2.23个等位基因;UPGMA聚类分析表明,15个六倍体燕麦种质资源在Dice系数为0.93处聚为3支,基本上是按照不同种进行聚类的,在相同种中又根据地理来源分别聚集成支。利用40对EST-SSR引物对31个遗传背景不清的燕麦种质资源进行基因组倍性鉴定,发现这些种质中可能存在有四倍体和二倍体的燕麦新资源。本研究开发的燕麦EST-SSR功能性标记将在燕麦遗传多样性分析、遗传图谱构建及燕麦属内种间基因组鉴定等方面发挥重要作用。     

Growth of three legume trees inoculated with VA mycorrhizal fungi and Rhizobium

Effectiveness among four VA mycorrhizal fungi and Rhizobium (R) in promoting growth of three legume trees in a P-deficient soil was studied.Glomus fasciculatus + R andGigaspora margarita + R were most effective forAcacia mangium andAlbizia falcataria (syn.:Paraserianthes falcataria). Scutellospora persica + R,Gigaspora margarita + R andGlomus fasciculatus + R were most effective forAcacia auriculifornis. Consistently poor growth was attained by seedlings inoculated withSclerocystis clavispora + R,Rhizobium alone, or by uninoculated seedlings.     

植物生长调节剂对黑木相思优树腋芽增殖及生根的影响

为建立黑木相思(Acacia melanoxylon)快繁技术体系,以含1个腋芽的无菌茎段为材料,研究了植物生长调节剂对其增殖和生根的影响。结果表明,6-BA 极易诱导黑木相思愈伤组织形成,但芽长势较差,不利于腋芽增殖体系的建立。而生长素既能诱导黑木相思生根,又能诱导腋芽增殖;将无菌茎段接入MS+IAA 0.5 mg L^-1+IBA 0.5 mg L^-1培养基中培养20 d的生根率为98.41%,培养40 d的腋芽增殖倍数为2.36,单株繁殖系数为6.57。这是首次成功建立高效、简便的生根和增殖同步发生的黑木相思直接器官发生途径的组培技术体系。     

Cross-amplification of nonnative Acacia species in the Hawaiian Islands using microsatellite markers from Acacia koa

Thirty-one microsatellite markers, developed from Acacia koa, were evaluated for cross-amplification of seven nonnative species of Acacia in the Hawaiian Islands. The number of alleles per locus ranged from one to seven, with 16 of the 31 markers showing polymorphism in at least two of the nonnative Acacia species. Of the Acacia species examined, A. melanoxylon showed the highest percentage of amplified loci.     

Development of EST-SSR markers and investigation of genetic relatedness in tung tree

The tung tree is an important non-edible oilseed source and consists of two species Vernicia fordii and Vernicia montana native to southern China and southeast Asia. In this study, the frequency and distribution of simple sequence repeats (SSRs) in expressed sequence tags (ESTs) of V. fordii were characterized based on 2,407 available EST sequences from the National Center for Biotechnology Information database. Twenty-two EST-SSR markers were developed by screening the genomic DNAs of six individuals of V. fordii from three accessions and 120 individuals of V. montana from 30 indigenous V. montana accessions collected from different geographic areas in southwestern China and northern Laos. The 22 EST-SSR markers exhibited a moderate level of polymorphism in V. montana with an average of 3.36 alleles per locus and PIC of 0.401. Genetic relatedness investigation showed that there was not only distinct genetic differentiation among tung trees but also a distinct geographic pattern among V. montana accessions. The current study is the first report of the development of EST-SSRs and genetic relatedness investigation in tung trees. These EST-SSR markers reported here will be valuable resources for future genetic studies, like construction of linkage maps, diversity analysis, quantitative trait locus/association mapping, and molecular breeding of the tung tree. The genetic relatedness identified in V. montana would provide potential clues in choosing germplasms in interest as progenitors for cross breeding and variety improvement of V. montana in practice.     

基于转录组测序的枫香EST-SSR引物开发及有效性评价

枫香是广西重要的乡土树种之一，具有较高的用材、观赏及药用价值。为验证枫香SSR引物的实际应用效果，该研究基于转录组测序技术，检测枫香SSR位点并设计引物，通过PCR扩增和聚丙烯酰胺凝胶电泳筛选出具有较高多态性的枫香EST-SSR引物，并对1个枫香天然群体进行遗传多样性分析。结果表明：(1)共发掘到23 777个SSR位点，单核苷酸重复类型SSR位点占总位点比例最高(46.54%),在重复次数上5～12次之间的SSR位点占比最高(72.36%)。(2)共开发出262对SSR引物，有效扩增率为53.1%,最终筛选出扩增稳定、条带清晰的引物18对。(3)多态性检测结果显示所有位点均具有多态性，天然群体遗传多样性结果显示该天然群体中等位基因数量(Na)、有效等位基因数量(Ne)、Shannon多样性指数(I)、观测杂合度(Ho)变化范围分别为2～4、1.112 8～2.609 6、0.208 9～1.112 7和0.275 9～1.000 0,平均值分别为2.333 3、1.957 4、0.708 5和0.722 6。综上认为，枫香中占优势的SSR位点重复类型和重复基序与其他物种基本相同，所开...     

Induction of nodule primordia on Phaseolus and Acacia by lipo-chitin oligosaccharide nodulation signals from broad-host-range Rhizobium strain GRH2

Rhizobium wild-type strain GRH2 was originally isolated from the tree, Acacia cyanophylla, and has a broad host-range which includes herbaceous legumes, such as Phaseolus and Trifolium species. Here we show that strains of Rhizobium sp. GRH2, into which heterologous nodD alleles have been introduced, produce a large diversity of both sulphated and non-sulphated lipo-chitin oligosaccharides (LCOs). Most of the molecular species contain an N-methyl group on the reducing-terminal N-acetyl-glucosamine. The LCOs vary in the nature of the fatty acyl chain and in the length of the chitin backbone. The majority of the LCOs have an olgosaccharide chain length of five GlcNAc residues, but a few are oligomers having six GlcNAc units. LCOs purified from GRH2 are able to induce root hair formation and deformation on Acacia cyanophylla and A. melanoxylon plants. We show that an N-vaccenoyl-chitopentaose bearing an N-methyl group is able to induce nodule primordia on Phaseolus vulgaris, A. cyanophylla, and A. melanoxylon, indicating that for these plants an N-methyl modification is sufficient for nodule primordia induction.     

Root surface area measurements based on adsorption and desorption of nitrite

A method, based on the negative adsorption of NO₂ ^-, has been developed to determine surface area of roots. Young roots of 3–4 year old plants ofAcacia catechu, Eucalyptus camaldulensis andLeucaena leucocephala were up-rooted and cut into 18 cylindrical pieces. Each root piece was immersed individually for 10 sec in 0.05M, 0.10M and 0.15M aqueous NaNO₂ solution and the excess solution on the root surface allowed to drain off. It was then transferred to conical flask containing distilled water and shaken for 15 min for desorption of nitrite. A known quantity of this aliquot was reacted with 1% acidic sulphanilamide and 0.02% NED HCl. A pink colour developed, and its optical density was read at 540 nm. A positive linear correlation was noted between colour density and root surface area. The respective correlation coefficientvalues for 0.05M, 0.10M and 0.15M NaNO₂ solutions were 0.973, 0.963 and 0.964 forAcacia catechu, 0.933, 0.903 and 0.898 forEucalyptus camaldulensis and 0.968, 0.976 and 0.972 forLeucaena leucocephala (significant atp<0.001). The method was successfully adopted to determine the root surface area of seedlings ofAlbizia lebbek, A. procera, Acacia auriculiformis, A. nilotica, Dalbergia latifolia andD. sissoo.     

Isolation and characterization of microsatellite loci of Lycopodium fordii Bak. (Lycopodiaceae, Pteridophyta)

Lycopodium fordii Bak. (Lycopodiaceae, Pteridophyta) is a gardening plant with a native, fragmentary distribution in Taiwan. In this study, we described the development of eleven microsatellite loci in L. fordii for genetic studies. These new markers were tested in 16 individuals of three populations. The number of alleles ranged from 1 to 5 and the expected heterozygosity from 0.41746 to 0.72222. Four of the nine polymorphic loci were significantly deviated from Hardy-Weinberg expectations due to the heterozygote deficiency. The microsatellite markers have also been proved as informative genetic markers for other 15 Lycopodium species.     

DNA typing and genetic relations among populations of Kelussia odoratissima using ISSR and SRAP markers

Kelussia odoratissima is well known for its medicinal importance. It has been announced as an endangered species. Thus, examining the genetic variation and conservation of this plant is necessary. In the present study, inter simple sequence repeat (ISSR) and sequence-related amplified polymorphism (SRAP) molecular markers were employed for the first time to access the genetic diversity and relationships of 77 wild individual plants of K. odoratissima collected from seven populations in Central Zagros region of Iran. A total of 146 bands were amplified by 12 ISSR primers, of which 129 (87.80 %) were polymorphic, while 69 polymorphic bands (83.30 %) were observed among 86 bands amplified by 11 SRAP primers. Polymorphic information content (PIC = 0.32), resolving power (Rp = 7.80), and marker informativeness (MI = 3.48) generated by ISSR primers were higher than that of SRAP analysis (PIC = 0.30, Rp = 5.61, and MI = 1.88). The study indicated that ISSR were more effective than SRAP markers for assessing the degree of genetic variation of K. odoratissima. In both UPGMA dendrograms of ISSR and SRAP, in most cases, individuals from each population were clustered in various groups without clear separation, which demonstrates the high variability of this germplasm in Iran. UPGMA cluster analysis revealed inconsistencies in the clustering patterns, as the Mantel’s test between the dendrograms for ISSR and SRAP data indicated a poor fit for the ISSR and SRAP data types (r = 0.10). Besides, principal coordinate analysis results showed that the first three principal coordinates account for 65.57 % of the total variation and studied seven populations were separated from each other and placed into five groups. These results have an important implication for K. odoratissima germplasm characterization, improvement, and conservation.     

黑木相思优良无性系叶片数量性状与生长评价

为合理利用种质资源,对5年生黑木相思(Acacia melanoxylon)无性系试验林中9个优良无性系(F1、SR3、SR9、SR14、SR17、SR18、SR25、SR38、SR41)开展叶片和生长性状的遗传变异研究,采用模糊数学隶属函数值法进行综合评价。结果表明,9个无性系叶片在形态、结构、化学性状都存在显著差异,以形态性状变异系数最大,其中叶面积(LA)和叶宽(LW)可作为直观区别无性系的可靠指标。SR18、SR17和SR14号无性系的树高(H)和胸径(DBH)生长具有显著优势。相关性分析表明,LW与H,LW、LA、比叶面积(SLA)与DBH呈显著线性关系,而叶长(LL)、干物质量(LDMC)、C、N、P、C/N和N/P与H和DBH相关性弱。隶属函数综合评价排名前4位的是SR17、SR18、SR41、SR14,可作为直接生产和后续育种工作的有效资源加以应用。