Matrix Generator(MG):一个基于DNA片段的0/1矩阵生成程序

分子群体遗传学研究的特点是取样量大--存在于群体样本中的遗传变异必须要充分代表该群体和该物种的遗传变异量及分析的位点数多--位点样本必须恰当代表基因组.大样本的群体取样和位点取样产生大量的原始数据,使原始数据人工处理非常困难甚至不可能,从而迫切需要原始数据处理的自动化.目前一些大公司提供的凝胶图像收集仪器和配套的软件已经使原始数据的获取基本上自动化或半自动化.获得DNA片段分子量数据后,必须把这些分子量数据转变成可反映操作单位(样本)之间关系的数据矩阵,原来用于计算分子量的那些软件已不实用或派不上用场.目前,除了用于fAFLP的Binthere弥补了部分不足外,还没有此类软件.Binthere存在固定栏宽(Bin)的缺陷,也就是将分子量最大值与最小值之间等分的方法来归纳不同操作单位(OUT)之间的异同,使得分子量绝对值差很小的数据可能被归入不同的栏,导致结果不正确.为了解决这类问题,我们设计编写了一个新的软件,取名为Matrix Generator(MG).与同类软件相比,MG具有两个主要优点:(1)采用动态栏宽和智能归并算法,克服了固定栏宽可能造成的错误;(2)可用于非荧光标记的分子标记技术.MG的基本思路是:分子量差异越小的片段,越可能是同缘片段,越应该处于相同的栏内.为此,我们采用绝对对应的动态过程.也就是说,从最小分子量到最大分子量之间的栏目数不是事先确定,而是由所分析的所有样品的特点和所使用的凝胶的分辨率(用户根据凝胶的特点给出数值)决定的.当两片段的差异小于凝胶所能达到的分辨率时,两片段被认为是同缘片段而归入相同的栏内.归并的过程从差异最小值开始,直至任意两片段的差异都大于凝胶的分辨率为止.这样就排除了同缘片段被隔离或者非同缘片段被合并的错误,从而使最可能同缘的片段归结在同一位点.MG第一版(V1.0,DOS版)集中体现了实用和易用的优点而没有包含同类软件所具有的一些功能,所以MG必须与其他软件结合使用.对于非荧光标记的分子标记技术,如RAPD、RFLP、AFLP等,可用Quantity One等软件得到分子量,用Excel生成样品与(分子量数据代表的)DNA片段矩阵,然后用MG处理.对于荧光标记的分子标记技术,如fAFLP、fSSR等,除可以用Excel生成矩阵外,可直接用Binthere和Genotyper等生成分子量矩阵,然后用MG处理.MG输出的矩阵经过适当编辑后,就可用后续的软件如Paup、Ntsys、Philip等运算.为了检验MG的有效性,我们用六道木属(Abelia)的AFLP分析数据进行检验,14个样品的DNA片段分别用Binthere和MG进行处理.前者得到295个含信息的位点,后者得到210个含信息的位点.用Nei and Li(1979)的算法分别计算距离矩阵并对两距离矩阵作Mantel检验.结果,两矩阵之间存在一定的差别,但相似性系数高达0.941 63,说明两种方法总体上会得到相似的结果,但局部会有所不同.用Paup对两矩阵作进一步分析,生成两个Neighbor-joining(NJ)树.结果表明,MG生成的数据更符合实际情况,而且分辨率高.     

微卫星座位对实验动物beagle犬的遗传分析

目的对美国进口、广州自养beagle犬基因组中存在的微卫星结构进行分析,研究其群体的微卫星多态性,以此探索在分子水平上对作为实验动物的beagle犬进行检测。方法通过微卫星分子标记技术进行遗传背景分析,并结合微卫星位点测序结果,研究DNA分子特征。结果在研究位点上共发现6个复等位基因,进口犬群体共有6个等位基因片段,自养犬群体共有5个等位基因片段,根据基因型计算各群体等位基因频率,由相关公式计算杂合度、群体多态信息含量(PIC)、基因纯合率、基因分化系数。结论两群体的杂合度、PIC值均较高(分别为0.7010、0.6747和0.7876、0.7515),基因分化系数很低(0.021),表明两群体没有形成明显的独立群。     

RFLP分子标记及其在牧草研究中的应用

RFLP分子标记是用限制性酶(RE)消化不同个体的同源DNA分子,经电泳表现的限制性片段长度的差异。它反映了DNA分子水平上的变异,可能是限制性内切酶识别位点的改变,也可能是部分片段的缺失、插入、易位、倒位等,显示出丰富的多态性。本文综述了RFLP分子标记技术在牧草个体识别、绘制遗传图谱、目的基因定位、检测群体内或群体间序列差异程度以及辅助育种研究中的应用等,并对该技术在牧草研究中的应用作了展望。     

微卫星标记在玉米群体遗传多样性研究中的应用

简要综述了近几年利用微卫星(SSR)分子标记技术分析玉米群体遗传多样性过程中的研究进展对研究中混合取样,荧光标记技术,共享数据库等新方法的使用作了阐述.     

染色体片段导入系在作物遗传育种中的应用

准确而有效的定位农作物数量性状基因座(Quantitative Trait Loci,QTLs)是植物分子育种的核心,传统的QTL定位群体遗传背景复杂,受群体大小和统计方法等多方面的限制,难以达到QTL精细定位。随着分子标记技术、计算机统计软件及分子辅助选择的飞速发展,一种新的QTL定位群体脱颖而出,这就是染色体片段导入系(Chromosome Segment Introgression Lines,CSILs)。它不但能有效消除"遗传背景噪音"对QTL定位的干扰,还能够在群体中挖掘出大量的有利隐蔽基因,对农作物遗传育种的进一步发展有巨大贡献。对染色体片段导入系的优越性,应用范围以及应用前景作以综述。     

基于转录组数据的齿缘刺猎蝽微卫星分子标记开发

【目的】齿缘刺猎蝽Sclomina erinacea是我国猎蝽科天敌昆虫常见种类,其不同地理种群存在明显形态差异。本研究旨在利用已经获得的齿缘刺猎蝽转录组数据筛选微卫星位点,为齿缘刺猎蝽种群遗传多样性和遗传分化研究开发可靠的微卫星分子标记。【方法】基于高通量测序技术平台Illumina HiSeq^TM 2000获得齿缘刺猎蝽转录组数据(42 215条unigenes),利用MISA软件进行搜索发掘SSR标记;利用Primer Premier 3软件设计SSR引物,从中随机选取54对SSR引物,利用PCR技术在中国齿缘刺猎蝽9个地理种群上进行验证。【结果】利用MISA软件搜索到微卫星位点2 395个,它们分布在2 107条unigenes上,其主要重复类型是三核苷酸重复(占总SSR的44.43%),其次是二核苷酸重复(占总SSR的40.08%),再次是四核苷酸重复(占总SSR的12.94%)。利用Primer Premier 3 软件成功设计出2 000余对SSR引物。随机选取的54对引物对9个齿缘刺猎蝽不同地理种群进行的SSR位点PCR扩增结果表明,共有16对引物较好地扩增出目的片段。【结论】研究表明利用齿缘刺猎蝽转录组数据可以大量发掘微卫星分子标记。本研究为进一步开展齿缘刺猎蝽的种群遗传学研究奠定了基础。     

我国沿海三疣梭子蟹9个野生群体线粒体CR和COⅠ片段比较分析

通过对我国沿海三疣梭子蟹(Portunus trituberculatus)9个野生群体线粒体DNA控制区(putative control region,CR)基因片段和细胞色素氧化酶亚基Ⅰ(COⅠ)基因片段进行扩增和测序,分别得到长度为530bp和584bp的片段。分析结果表明:CR83条序列A T平均含量为73·2%,共检测到91个变异位点,83个个体具有66种单倍型(haplotype);COⅠ94条序列A T平均含量为62·2%,共检测到34个变异位点,94个个体具有34种单倍型(haplotype)。用MEGA3·1软件构建9个群体NJ分子树,聚类结果显示,黄海、东海、渤海6个群体之间的相对遗传距离比较近,聚为一大支,南海3个群体相对遗传距离比较近,聚为一大支。AMOVA分析表明,群体间的遗传分化指数(Fst)为-0·07454~0·27087,其中部分群体间达到显著差异(P<0·05)与极显著差异(P<0·01),说明我国三疣梭子蟹不同野生群体间存在一定遗传分化。     

小麦高分子量谷蛋白基因转录调控相关的顺反式相互作用的初步研究

利用凝胶延滞（ｇｅｌｒｅｔａｒｄａｔｉｏｎ）分析技术, 研究了小麦（Ｔｒｉｔｉｃｕｍ ａｅｓｔｉｖｕｍ ）高分子量谷蛋白基因转录启始点上游９００ ｂｐ 的ＤＮＡ 片段与未成熟小麦胚乳细胞核因子的顺反式相互作用。利用ＤＮＡ聚合酶链反应技术, 将９００ ｂｐ 分成４ 段凝胶延滞分析用探针, 并以高盐浓度抽提法从扬花１２ ｄ 左右的小麦胚乳得到了核抽提物。凝胶延滞分析结果表明, 这４ 个ＤＮＡ片段上均存在与各自核蛋白因子专一性结合的位点。推测该基因的表达是受多位点复杂的顺反式相互作用来调控的     

辽东湾斑海豹遗传多样性的AFLP分析

利用AFLP标记技术对辽东湾斑海豹的遗传多样性进行分析。采用7对AFLP引物对斑海豹3个群体(按不同采样年份分类)43个个体扩增共得到241个位点,3个群体内的多态位点比例为80.45%～95.85%,总多态位点比例为99.59%。群体的香农(Shannon)多样性指数为0.3817～0.4716,群体间的遗传距离在0.1742～0.4023之间。2007年群体的多态位点比例、Shannon多样性指数均高于2006年群体,2005年群体处于中等水平。用NTSYS软件进行个体聚类及UPGMA方法构建的群体系统树,发现3个群体的个体基本随机聚到一起,界限不十分明显,说明3年的群体差异不明显,甚至可以认为它们是混合群体。结果表明,斑海豹群体遗传多样性水平较低,遗传结构趋于简单化,且存在较强的基因交流。     

三角帆蚌五个野生群体线粒体DNA 16S rRNA遗传特性

对中国主要淡水湖泊(鄱阳湖、洞庭湖、太湖、洪泽湖及巢湖)三角帆蚌5个野生群体的线粒体DNA 16S rRNA基因片段进行了扩增和测序,得到473bp的碱基序列,没有发现插入/缺失突变的核苷酸位点。检测到了32个多态性核苷酸位点,共7种单倍型。鄱阳湖群体的222(C→G)和325(A→G)位点,太湖群体的233(A→G)位点,巢湖群体的40(A→G)、138(A→T)和294(C→T)位点,洪泽湖群体的241(A→C)位点的变异可以作为区分群体分子遗传标记位点。洞庭湖群体未发现特异位点。在5个群体间鄱阳湖群体多态性位点、核苷酸多态性、单倍型多态性和单倍型数量4个指标都最高,表明鄱阳湖群体具有最为丰富的遗传结构,遗传变异最大,可作为三角帆蚌选育的基础群体。     

Physical and genetic map of the Lactococcus lactis subsp. cremoris MG1363 chromosome: comparison with that of Lactococcus lactis subsp. lactis IL 1403 reveals a large genome inversion.

A physical and genetic map of the chromosome of the Lactococcus lactis subsp. cremoris reference strain MG1363 was established. The physical map was constructed for NotI, ApaI, and SmaI enzymes by using a strategy that combines creation of new rare restriction sites by the random-integration vector pRL1 and ordering of restriction fragments by indirect end-labeling experiments. The MG1363 chromosome appeared to be circular and 2,560 kb long. Seventy-seven chromosomal markers were located on the physical map by hybridization experiments. Integration via homologous recombination of pRC1-derived plasmids allowed a more precise location of some lactococcal genes and determination of their orientation on the chromosome. The MG1363 chromosome contains six rRNA operons; five are clustered within 15% of the chromosome and transcribed in the same direction. Comparison of the L. lactis subsp. cremoris MG1363 physical map with those of the two L. lactis subsp. lactis strains IL1403 and DL11 revealed a high degree of restriction polymorphism. At the genetic organization level, despite an overall conservation of gene organization, strain MG1363 presents a large inversion of half of the genome in the region containing the rRNA operons.     

Utilization of unconventional lignocellulosic waste biomass for the biosorption of toxic triphenylmethane dye malachite green from aqueous solution

Biosorption potential of novel lignocellulosic biosorbents Musa sp. peel (MSP) and Aegle marmelos shell (AMS) was investigated for the removal of toxic triphenylmethane dye malachite green (MG), from aqueous solution. Batch experiments were performed to study the biosorption characteristics of malachite green onto lignocellulosic biosorbents as a function of initial solution pH, initial malachite green concentration, biosorbents dosage, and temperature. Biosorption equilibrium data were fitted to two and three parameters isotherm models. Three-parameter isotherm models better described the equilibrium data. The maximum monolayer biosorption capacities obtained using the Langmuir model for MG removal using MSP and AMS was 47.61 and 18.86 mg/g, respectively. The biosorption kinetic data were analyzed using pseudo-first-order, pseudo-second-order, Elovich and intraparticle diffusion models. The pseudo-second-order kinetic model best fitted the experimental data, indicated the MG biosorption using MSP and AMS as chemisorption process. The removal of MG using AMS was found as highly dependent on the process temperature. The removal efficiency of MG showed declined effect at the higher concentrations of NaCl and CaCl₂. The regeneration test of the biosorbents toward MG removal was successful up to three cycles.     

Restriction analysis of Thiobacillus ferrooxidans DNA using pulsed field electrophoresis]

The DNA of Thiobacillus ferrooxidans digested by any of the five restriction endonucleases (DraI, EcoRI, Eco321, HindIII, XbaI) was studied by electrophoresis in the pulsating differently directed electric fields (PF). The influence of PF conditions on the sized row of the divided DNA fragments was studied. Only the XbaI restriction endonuclease was shown to cleave the Thiobacillus ferrooxidans DNA into a number of fragments permitting one to define the sizes of fragments and genome (no more than 2300 bp). The prospects of using the restriction analysis of Thiobacillus ferrooxidans wild type culture for improving its properties in obtaining heavy metals are discussed.     

SfiI genomic cleavage map of Escherichia coli K-12 strain MG1655.

An SfiI restriction map of Escherichia coli K-12 strain MG1655 is presented. The map contains thirty-one cleavage sites separating fragments ranging in size from 407 kb to 3.7 kb. Several techniques were used in the construction of this map, including CHEF pulsed field gel electrophoresis; physical analysis of a set of twenty-six auxotrophic transposon insertions; correlation with the restriction map of Kohara and coworkers using the commercially available E. coli Gene Mapping Membranes; analysis of publicly available sequence information; and correlation of the above data with the combined genetic and physical map developed by Rudd, et al. The combination of these techniques has yielded a map in which all but one site can be localized within a range of +/- 2 kb, and over half the sites can be localized precisely by sequence data. Two sites present in the EcoSeq5 sequence database are not cleaved in MG1655 and four sites are noted to be sensitive to methylation by the dcm methylase. This map, combined with the NotI physical map of MG1655, can aid in the rapid, precise mapping of several different types of genetic alterations, including transposon mediated mutations and other insertions, inversions, deletions and duplications.     

Differences in seed rain composition in small and large fragments in the northeast Brazilian Atlantic Forest

Tropical forests are seriously threatened by fragmentation and habitat loss. The impact of fragment size and forest configuration on the composition of seed rain is insufficiently studied. For the present study, seed rain composition of small and large forest fragments (8–388 ha) was assessed in order to identify variations in seed abundance, species richness, seed size and dispersal mode. Seed rain was documented during a 1‐year period in three large and four small Atlantic Forest fragments that are isolated by a sugarcane matrix. Total seed rain included 20,518 seeds of 149 species of trees, shrubs, palms, lianas and herbs. Most species and seeds were animal‐dispersed. A significant difference in the proportion of seeds and species within different categories of seed size was found between small and large fragments. Small fragments received significantly more very small‐sized seeds (<0.3 cm) and less large‐seeded species (>1.5 cm) that were generally very rare, with only one species in small and eight in large fragments. We found a negative correlation between the inflow of small‐sized seeds and the percentage of forest cover. Species richness was lower in small than in large fragments, but the difference was not very pronounced. Given our results, we propose changing plant species pools through logging, tree mortality and a high inflow of pioneer species and lianas, especially in small forest fragments and areas with low forest cover. Connecting forest fragments through corridors and reforestation with local large‐seeded tree species may facilitate the maintenance of species diversity.     

Campbell-like integration of heterologous plasmid DNA into the chromosome of Lactococcus lactis subsp. lactis

Integrable vectors were constructed based on the plasmid pHV60, which is essentially a pBR322 replicon carrying a chloramphenicol resistance marker, by inserting 1.3-kilobase chromosomal fragments of Lactococcus lactis subsp. lactis MG1363 into this plasmid. Three constructs as well as pHV60 were electroporated to strain MG1363. Transformants were obtained with all constructs, and also with pHV60 (albeit with low frequency). By using Southern hybridizations, it appeared that pHV60 showed homology with the chromosome of MG1363, and that it most probably uses this homology to integrate in a Campbell-like manner. The presence of chromosomal sequences in pHV60 stimulated insertion elsewhere in the chromosome by a factor of 5 to 100. In all cases the integrated plasmids were amplified, at a selective pressure of 5 micrograms of chloramphenicol per ml, to a level of approximately 15 copies per chromosome. Although the amplification was gradually lost under nonselective conditions, one copy remained stably integrated in the chromosome. The results show that a Campbell-like integration strategy can be used to improve the accessibility of the lactococcal chromosome for genetic analysis and is potentially useful in stabilizing unstable genes in lactococci.     

Biosorption of Malachite Green from aqueous solutions onto aerobic granules: kinetic and equilibrium studies

Batch experiments were conducted to study the biosorption characteristics of a cationic dye, Malachite Green (MG), onto aerobic granules. Effects of pH, aerobic granule dosage, contact time and solution temperature on MG biosorption by aerobic granules were evaluated. Simultaneity the thermodynamic analysis was also performed. The results showed that alkaline pH was favorable for the biosorption of MG and chemisorption seemed to play a major role in the biosorption process. Kinetic studies indicate that MG biosorption on aerobic granules in the system follows the pseudo-second order kinetics. The equilibrium time was 60 min for both 50 and 60 mg/L and 120 min for both 70 and 80 mg/L MG concentrations, respectively. Moreover, the experimental equilibrium data have been analyzed using the linearized forms of Langmuir, Freundlich, and Redlich-Peterson isotherms and the Langmuir isotherm was found to provide the best theoretical correlation of the experimental data for the biosorption of MG. The monolayer biosorption (saturation) capacities were determined to be 56.8 mg of MG per gram of aerobic granules at 30 degrees C. Thermodynamic analysis show that biosorption follows an endothermic path of the positive value of Delta H( composite function) and spontaneous with negative value of Delta G( composite function).     

Campbell-like integration of heterologous plasmid DNA into the chromosome of Lactococcus lactis subsp. lactis.

Integrable vectors were constructed based on the plasmid pHV60, which is essentially a pBR322 replicon carrying a chloramphenicol resistance marker, by inserting 1.3-kilobase chromosomal fragments of Lactococcus lactis subsp. lactis MG1363 into this plasmid. Three constructs as well as pHV60 were electroporated to strain MG1363. Transformants were obtained with all constructs, and also with pHV60 (albeit with low frequency). By using Southern hybridizations, it appeared that pHV60 showed homology with the chromosome of MG1363, and that it most probably uses this homology to integrate in a Campbell-like manner. The presence of chromosomal sequences in pHV60 stimulated insertion elsewhere in the chromosome by a factor of 5 to 100. In all cases the integrated plasmids were amplified, at a selective pressure of 5 micrograms of chloramphenicol per ml, to a level of approximately 15 copies per chromosome. Although the amplification was gradually lost under nonselective conditions, one copy remained stably integrated in the chromosome. The results show that a Campbell-like integration strategy can be used to improve the accessibility of the lactococcal chromosome for genetic analysis and is potentially useful in stabilizing unstable genes in lactococci.     

The development of mitochondrial membrane affinity chromatography columns for the study of mitochondrial transmembrane proteins

Mitochondrial membrane fragments from U-87 MG (U87MG) and HEK-293 cells were successfully immobilized onto immobilized artificial membrane (IAM) chromatographic support and surface of activated open tubular (OT) silica capillary, resulting in mitochondrial membrane affinity chromatography (MMAC) columns. Translocator protein (TSPO), located in mitochondrial outer membrane as well as sulfonylurea and mitochondrial permeability transition pore (mPTP) receptors, localized to the inner membrane, were characterized. Frontal displacement experiments with multiple concentrations of dipyridamole (DIPY) and PK-11195 were run on MMAC (U87MG) column, and the binding affinities (K_d) determined were 1.08 ± 0.49 and 0.0086 ± 0.0006 μM, respectively, consistent with previously reported values. Furthermore, binding affinities (K_i) for DIPY binding site were determined for TSPO ligands, PK-11195, mesoporphyrin IX, protoporphyrin IX, and rotenone. In addition, the relative ranking of these TSPO ligands based on single displacement studies using DIPY as marker on MMAC (U87MG) was consistent with the obtained K_i values. The immobilization of mitochondrial membrane fragments was also confirmed by confocal microscopy.     

GenoProfiler: batch processing of high-throughput capillary fingerprinting data

High-throughput content fingerprinting techniques employing capillary electrophoresis place new demands on the editing of fingerprint files for the downstream contig assembly program, FPC. A cross-platform software application, GenoProfiler, was developed for automated editing of sized fingerprinting profiles generated by the ABI Genetic Analyzers. The batch-processing module extracts the sized fragment information directly from the ABI raw trace files, or from data files exported from GeneMapper or other size calling software, removes the background noise and undesired fragments, and generates fragment size files compatible with the FPC software. AVAILABILITY: http://wheat.pw.usda.gov/PhysicalMapping/