基于In-fusion技术的ERECTA基因植物表达载体的构建

以黄瓜品种‘长春密刺’(CCMC)为试材,利用高保真酶Iproof及不依赖于酶切的In-fusion技术,构建基于本底启动子驱动的黄瓜ERECTA基因的植物表达载体,以探讨ERECTA基因在黄瓜中的功能,以期为黄瓜抗性性状的遗传改良提供技术支持。结果表明:从CCMC的基因组DNA中克隆的2段gERECTA(DNA序列)长度分别为8 940bp和9 812bp,并分别命名为CsgERECTA-Flag和CsgERECTA-Poly。经过质粒PCR、梯度片段PCR和测序结果的鉴定表明,pCAMBIA3301-gERECTA的2个植物表达载体都已成功构建并转入根癌农杆菌EHA105中。

利用AFLP指纹技术研究中国沿海真鲷群体的遗传变异和趋异

用AFLP指纹技术对中国沿海真鲷群体进行遗传变异和趋异分析 ,使用了 6对引物 :E -ACC/M -CAT、E -AGG/M -CTG、E -AGG/M -CTT、E -AAC/M -CTA、E -ACA/M -CTG、E -AAC/M -CAA ,在威海、厦门和北部湾海区三个野生群体共 42个体中检出了 6 46个扩增位点 (片段大小 5 0～ 40 0bp) ,其中 70 %位点在群体内或群体间呈现多态。三个群体的多态位点比例变化在 5 8.4%～ 6 4.0 % ,遗传相似系数变化在 0 .842 5～0 .82 0 0 ,以威海群体的遗传变异量最大 ,北部湾群体的最小。三群体间遗传距离和UPGMA谱系图显示 ,厦门海区与北部湾的真鲷差异较小 ,可以认为是同一个亚种群的不同群体 ,黄海真鲷与前二个群体差异比较大 ,属于另一个不同的亚种群。

Discussion on AFLP Molecular Markers in Piper methysticum and Pepper

The aim of the research was to discuss the genetic relationships between Piper methysticum, Pepper and other wild species in Pepper genus. DNA was extracted from leaves which belonged to 28 germplasms including 6 materials of P. methysticum, 21 maerials of cultivated and wild Pepper, 1 material of Peperomia pellucida belonged to different genus. Premiers with good band-type and high polymorphism and resolution were selected from 64 pairs of primers for AFLP amplification and the clustering analysis was conducted with MVSP3.13f software. 191 bands were amplified by 4 pairs of premiers, 189 of which had polymorphism, being 98.6%. 28 germplasms were classified into 6 different groups at the genetic similarity coefficient of 0.52 by silver staining AFLP, in which 6 materials of Piper methysticum were clustered into a single group, indicating that P. methysticum belonged to Pepper family of Pepper genus but were distantly related to the others. The research provided the basis for selecting rootstocks for P. methysticum graft, molecular identification of P. methysticum and the fingerprint construction of P. methysticum.

A genetic linkage map of marine shrimp Penaeus (Fenneropenaeus) chinensis based on AFLP, SSR, and RAPD markers

The Chinese shrimp Penaeus (Fenneropaeneus) chinensis is an important species in marine fishery and aquaculture in China. A female Chinese shrimp Penaeus (Fenneropaeneus) chinensis was captured from west coast of the Korean peninsula and mated with a "Yellow Sea No. 1" male to produce the first filial generation (F1) 100 F2 full-sib progeny from brother-sister crosses between F1 families was used for the mapping study. A genetic linkage map of the Chinese shrimp was constructed, based on 354 markers, including 300 amplified fragment length polymorphism (AFLP) markers, 42 microsatellite (SSR) markers, and 12 randomly amplified polymorphism (RAPD) markers. Forty-seven linkage groups (LGs) were identified. The total map length was 4 580.5 cM, with an average spacing of 11.3 cM, covering 75.8% of the estimated genome size. The construction of this genetic linkage map was part of a genetic breeding program. This linkage map will contribute to the discovery of genes and quantitative trait loci (QTLs) in Chinese shrimp.

Genetics and phylogeny of genus Coilia in China based on AFLP markers

The taxonomy of Coilia has been extensively studied in China,and yet phylogenetic relationships among component taxa remain controversial.We used a PCR-based fingerprinting technique,amplified fragment length polymorphism(AFLP) to characterize and identify all four species of Coilia in China.We examined the genetic relationships of the four species of Coilia and a subspecies of Coilia nasus with AFLP.A total of 180 AFLP loci were generated from six primer combinations,of which 76.11% were polymorphic.The mean genetic distance between pairs of taxa ranged from 0.047 to 0.596.The neighbor-joining tree and UPGMA dendrogram resolved the investigated species into three separate lineages:(1) C.mystus,(2) C.grayii and(3) C.brachygnathus,C.nasus,and C.nasus taihuensis.Phylogenetic analysis of the AFLP data is inconsistent with current morphological taxonomic systems.The AFLP data indicated a close relationship among C.brachygnathus,C.nasus taihuensis,and C.nasus.Therefore,the two species described under Coilia(C.brachygnathus and C.nasus taihuensis) are treated as synonyms of C.nasus.

Genetic Variation and Differentiation in Wide Ranging Populations of Razor Clam(Sinonovacula constricta) Inferred from AFLP Markers

The genetic variation and differentiation of the razor clam Sinonovacula constricta distributed along the coast of China were studied through amplified fragment length polymorphism(AFLP) analysis.Six primer combinations generated 193 fragments.The He values varied from 0.322 to 0.463 and the percentage of polymorphic loci ranged from 74.1% to 98.4%,which indicates a high level of genetic diversity.Cluster analysis by Nei's pairwise distance grouped all specimens by geographical origins.AMOVA consistently showed that genetic variation among populations was 8.71%,and most of the variation came from the genetic variation within populations(91.29%).Genetic differentiation among the six populations was moderate;pairwise FST ranged from 0.0282 to 0.1480,which indicated that S.constricta populations along the coast of China are genetically connected.Among all the six populations,the Beihai population is the mostly differentiated from the others,suggesting that Hainan Island and Leizhou Peninsula act as barriers to gene flow.All populations abide isolation by distance model as indicated by Mantel test,except for ZS(Zhoushan) and YQ(Yueqing) populations.Information obtained in this study will provide guidelines for conservation and fishery management of this species in the future.

Genetic diversity of populations and clones of Rhopilema esculentum in China based on AFLP analysis

Amplified fragment length polymorphisms(AFLP) markers were developed to assess the genetic variation of populations and clones of Rhopilema esculentum Kishinouye(Scyphozoa,Rhizostomatidae).One hundred and seventy-nine loci from 56 individuals of two hatchery populations and two wild populations were genotyped with five primer combinations.The polymorphic ratio,Shannon's diversity index and average heterozygosity were 70.3%,0.346 and 0.228 for the white hatchery population,74.3%,0.313,and 0.201 for the red hatchery population,79.3%,0.349,and 0.224 for the Jiangsu wild population,and 74.9%,0.328 and 0.210 for the Penglai wild population,respectively.Thus,all populations had a relatively high level of genetic diversity.A specific band was identified that could separate the white from the red hatchery population.There was 84.85% genetic differentiation within populations.Individual cluster analysis using unweighted pair-group method with arithmetic mean(UPGMA) suggested that hatchery populations and wild populations could be divided.For the hatchery populations,the white and red populations clustered separately;however,for the wild populations,Penglai and Jiangsu populations clustered together.The genetic diversity at the clone level was also determined.Our data suggest that there are relatively high genetic diversities within populations but low genetic differentiation between populations,which may be related to the long-term use of germplasm resources from Jiangsu Province for artificial seeding and releasing.These findings will benefit the artificial seeding and conservation of the germplasm resources.

Application of AFLP markers for population genetic study on half-smooth tongue sole Cynoglossus semilaevis

The genetic diversity of wild and hatchery populations of half-smooth tongue sole Cynoglossus semilaevis, based on observation of amplified fragment length polymorphism (AFLP) was described. Two hundred individuals from four wild populations, Laizhou (LZ), Weihai (WH), Qingdao (QD), Rizhao (RZ), and one hatchery population, Mingbo (MB), were screened using eight different AFLP primer combinations. A total of 384 loci were screened in the five studied populations. 48.4%, 51.3%, 50.7%, 49.3% and 45.8% of these loci were polymorphic among the individuals tested in the LZ, WH, QD, RZ and MB populations, respectively. The number of polymorphic loci detected by single primer combinations ranged from 17 to 35. The average heterozygosity of the LZ, WH, QD, RZ and MB populations was 0.072, 0.093, 0.092, 0.090 and 0.063, respectively. The WH population showed the highest genetic diversity in terms of total number of AFLP bands, total number of polymorphic bands, average heterozygosity and percentage of low frequency (0-0.2) polymorphic loci among all the populations, while the LZ population was the lowest among the wild populations. Compared with the wild populations, the hatchery population showed a low genetic viability.

Genetic Analysis of Floating Enteromorpha prolifera in the Yellow Sea with AFLP Marker

Extremely large accumulation of green algae Enteromorpha prolifera floated along China'coastal region of the Yellow Sea ever since the summer of 2008.Amplified Fragment Length Polymorphism (AFLP) analysis was applied to assess the genetic diversity and relationships among E. prolifera samples collected from 9 affected areas of the Yellow Sea.Two hundred reproducible fragments were generated with 8 AFLP primer combinations,of which 194 (97%) were polymorphic. The average Nei's genetic diversity, the coefficiency of genetic differentiation (Gst), and the average gene flow estimated from Gst in the 9 populations were 0.4018, 0.6404 and 0.2807 respectively. Cluster analysis based on the unweighed pair group method with arithmetic averages (UPGMA) showed that the genetic relationships within one population or among different populations were all related to their collecting locations and sampling time. Large genetic differentiation was detected among the populations.The E. prolifera originated from different areas and were undergoing a course of mixing.

Preliminary Analysis of Population Genetic Diversity of Cultivated Laminaria japonica Sporophyte via AFLP Technique

The amplified fragment length polymorphic DNA (AFLP) technique was adopted to estimate the population genetic polymorphism among 30 sporophytes of Laminaria japonica collected from a cultivating farm in Rongcheng,China.Three methods were used for genomic DNA extraction from Laminaria japonica sporophyte and only the products obtained using the improved genomic DNA extraction kit method proved qualified for AFLP analysis.The parameters of the method were optimized.Samples of forty milligrams and the cell lysis time of 120 min were suggested to replace the parameters recommended by the manufacturer.Thirty individuals of Laminaria japonica from the same cultivating site were investigated using one pair of selective primers.A total of 21 loci were obtained and 17 of them were polymorphic.The mean percent age of polymorphic loci of this population was 80.95%.The Nei's gene diversity (H) within this population was 0.3028 and the average Shannon's Information index (I) was 0.4498.A genetic distance matrix among different individuals was constructed as well.Through this study,an applicable AFLP genetic analysis working system for Laminaria japonica sporophyte was established.The results of this research also revealed a high level of genetic diversity within the studied population.

Discriminating ability of molecular markers and morphological characterization in the establishment of genetic relationships in cultivated genotypes of almond and related wild species

A total 23 morphological traits, 19 AFLP-primer combinations, 80 RAPD primers and 32 SSR primer pair were used to compare the informativeness and efficiency of random amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP) and simple sequence repeat (SSR) markers in establishing genetic relationships among 29 almond cultivars and three related wild species. SSRs presented a high level of polymorphism and greater information content, as assessed by the expected hetrozygosity, compared to AFLPs and RAPDs. The lowest values of expected hetrozygosity were obtained for AFLPs; however AFLPs showed the highest efficiency, owing to their capacity to reveal large numbers of bands per reaction, which led to high values for various types of indices of diversity. All the three techniques discriminated almond genotypes very effectively, except that SSRs failed to discriminate between ‘Monagha’ and ‘Sefied’ almond genotypes. The correlation coefficients of similarity were statistically significant for all the three marker systems, but were lower for the SSR data than for RAPDs and AFLPs. For all the markers, high similarity in dendrogram topologies was obtained, although some differences were observed. All the dendrograms, including that obtained by the combined use of all the marker data, reflect relationships for most of cultivars according to their geographic diffusion. AMOVA detected more variation among cultivated and related wild species of almond within each geographic group. Bootstrap analysis revealed that the number of markers used was sufficient for reliable estimation of genetic similarity and for meaningful comparisons of marker types.

Genetic Diversity Analysis of Cultured Populations of Jade Perch （Scortum bacoo） in China Using AFLP Markers

Genetic diversity of three cultured populations of jade perch （Scortum bacoo） are studied using amplified fragment length polymorphism （AFLP） markers, which is Guangzhou（GZ） population, Foshan （FS） population and Qingdao（QD） population. Nine primer combinations were used and 385 fragments were detected. Among the 385 fragments, 80 bands （20.78%） were polymorphic. And it can be speculated that the genetic diversity of the three cultured populations of jade perch was very poor according to the gene genetic diversity among populations （Ht）, the gene genetic diversity within populations （Hs） and Shannon-Weiner index （I）. From gene differentiation （Gst）, genetic distance （D）, genetic similarity （5）, and UPGMA analysis, it is found that the relationship among the three populations is very closed, and the difference in genetic diversity among the three populations is not significant. Further clustering relationships of the jade perch cultured populations also are correlated to historical-breeding observations and genetic relationships. The GZ population clustered together with the QD population first, then with FS population.

AFLP analysis of genetic variation among three natural populations of horseshoe crab Tachypleus tridentatus along Chinese coast

The AFLP(amplified fragment length polymorphism) technique was used to analyze and compare the genetic diversity of Tachypleus tridentatus from three south-eastern coastal sites of China(Pingtan,Hong Kong and Beihai).Eight pairs of primers generated 361 loci,including 285 polymorphic loci.The ratio of polymorphic loci was 96.97%.Nei's genetic diversity index was 0.420 8 and the Shannon information index was 0.607 5,both of which were higher than that reported for many other arthropods.These results show that the genetic diversity detected was mainly caused by individual differences within a population.Genetic distance showed that the rational division of the three geographic populations of T.tridentatus along the south-eastern coast of China was not significant,in which the genetic distance was not proportional to the geographic distance.All three horseshoe crab populations may belong to a large group,and had a high degree of genetic similarity.The high level of genetic diversity obtained from the present AFLP analysis may be due to the large effective population size of the species in Chinese waters.

Microwave induced mutagenesis of Lactobacillus casei subsp. rhamnosus for enhancing L-lactic acid production

Mutagenesis of Lactobacillus casei subsp, rhamnosus Xl-12 after low power microwave irradiation was investigated. Under a microwave power of 400 W and irradiation length of 3 min, a mutated strain W4-3-9 with high-yield L-lactic acid was obtained by screening. Compared with the starting strain X1-12, the L-lactic acid production of W4-3-9 was increased by 58.0% at a concentration of 115.8 g/L. The strain maintained the capability of producing a high L-lactic acid level after 10 generations. Cell surface morphology and DNA structures of parental and mutated strains were observed by atomic force microscopy （ AFM ）. Amplified fragment length polymorphism （AFLP） analysis suggested the difference in AFLP band pattern between the mutated and non-mutated strains. Sequencing and BLAST analysis revealed that the catalytic site of lactate dehydrogenase （DHL） was changed due to the microwave induced mutation.

Significant population genetic structure detected in the rock bream Oplegnathus fasciatus(Temminck & Schlegel,1844) inferred from fluorescent-AFLP analysis

Oplegnathusfasciatus （rock bream） is a commercial rocky reef fish species in East Asia that has been considered for aquaculture. We estimated the population genetic diversity and population structure of the species along the coastal waters of China using fluorescent-amplifed fragment length polymorphisms technology. Using 53 individuals from three populations and four pairs of selective primers, we amplified 1 264 bands, 98.73% of which were polymorphic. The Zhoushan population showed the highest Nei＇s genetic diversity and Shannon genetic diversity. The results of analysis of molecular variance （AMOVA） showed that 59.55% of genetic variation existed among populations and 40.45% occurred within populations, which indicated that a significant population genetic structure existed in the species. The pairwise fixation index Fst ranged from 0.20 to 0.63 and were significant after sequential Bonferroni correction. The topology of an unweighted pair group method with arithmetic mean tree showed two significant genealogical branches corresponding to the sampling locations of North and South China. The AMOVA and STRUCTURE analyses suggested that the O.fasciatus populations examined should comprise two stocks.

Genomic and proteomic analysis of soybean heritable variations induced by space flight

To analyze the biological effects of space environment,the diversity of genomic DNA between the space flight soybean 194(4126) with phenotype of good yield and good fruit quality induced by space flight and the soybean with ground control was studied by amplified fragment length polymorphism(AFLP) method,and the polymorphism of space flight soybean 194(4126) was 3.56%.The differences of protein expression of seeds and leaves between the two kinds of soybeans were analysed by two-dimensional electrophoresis,PDQuest software and MALDI-TOF mass spectrometry.Results show that the loss and decrease of protein expression in 194(4126) soybean are subjected to the space fight of seeds,and three special proteins including Dehydrin,MAT1 and ceQORH are identified.It is concluded that the space environment changes the phenotype and genotype of soybeans due to the space flight of seeds.

Preliminary genetic linkage map of the abalone Haliotis diversicolor Reeve

Haliotis diversicolor Reeve is one of the most important mollusks cultured in South China. Preliminary genetic linkage maps were constructed with amplified fragment length polymorphism (AFLP) markers. A total of 2 596 AFLP markers were obtained from 28 primer combinations in two parents and 78 offsprings. Among them, 412 markers (15.9%) were polymorphic and segregated in the mapping family. Chi-square tests showed that 151 (84.4%) markers segregated according to the expected 1:1 Mendelian ratio (P<0.05) in the female parent, and 200 (85.8%) in the male parent. For the female map, 179 markers were used for linkage analysis and 90 markers were assigned to 17 linkage groups with an average interval length of 25.7 cm. For the male map, 233 markers were used and 94 were mapped into 18 linkage groups, with an average interval of 25.0 cm. The estimated genome length was 2 773.0 cm for the female and 2 817.1 cm for the male map. The observed length of the linkage map was 1 875.2 cm and 1 896.5 cm for the female and male maps, respectively. When doublets were considered, the map length increased to 2 152.8 cm for the female and 2 032.7 cm for the male map, corresponding to genome coverage of 77.6% and 72.2%, respectively.

Identification and Diagnostics of Plant-Symbiotic and Phytopathogenic Bacteria

Rapid and reliable diagnostics and identification of pathogenic and symbiotic bacteria are at the top of the agenda. In the first case, they are important to control and prevent crop damages, and thus reduce economic losses. In the second, it＇s necessary to design and monitor quality of biofertilizer to raise its effectiveness and crop capacity. Development of accurately, rapidly, technically and commercially accessible methods remains a critical problem for the bacteria with comprehensive phylogenetic structure. In this work, we investigated pathogenic Xanthomonas and Ralstonia and symbiotic Sinorhizobium. The aim of this investigation was to examine the applicability of the novel methods for phylogenetic study, identification and diagnostics of closely related species of these genera. The conventional phenotypic and genotypic （16S rRNA, gyrB） methods were applied as referents. Novel polymerase chain reaction （PCR）-based approaches, single-adapter amplified fragment length polymorphism （saAFLP） and comparative analyses of hin-region and Xcc0006-0007 sequences, were first employed for the investigations. Phenotypic tests, 16S rRNA and gyrB analysis distinguished bacteria at the genus level, but failed to identify them to the species robustly. The new methods identified bacteria at the inter-species level more precisely. This identification agreed with the accepted genera＇s classifications. The only exceptions were X. fuscans ＆ X. cirri and X. perforance ＆ X. euvesicatoria which clustered together. The further outcome of this study was achieved hin-region-based genus-specific PCR primers for the express-diagnostics of the genera. Summary, these new methods can be applied for genome-based phylogeny investigations and as convenient and accurate tools for identification and routine laboratory diagnostics of these comprehensive genera.

Genetic Diversity of the Medicinal Plant Ocimum Gratissimum L. （mint） from Kenya Based on AFLP Markers

Ocimum gratissimum L. native to Africa is a shrubby essential oil containing plant with medicinal, antimicrobial and antihelminthic properties. It is an important herbal medicine not only among Kenyan communities but also in the sub-Saharan Africa. No systematic assessment of genetic variability in O. gratissimum of Kenya has been carried out. In this study, amplified fragment length polymorphism （AFLP） analysis was used to estimate genetic diversity and genetic differentiation in 139 samples from all the different provinces of Kenya. Seven primer pairs, the Eco＋ACT plus Mse＋CAA, Eco＋ACT plus Mse＋CTG, Eco＋ACA plus Mse＋CAA, Eco＋ACA plus Mse＋CTG, Eco＋AGC plus Mse＋CAA, Eco＋ACC plus Mse＋CAA, and Eco＋ACC plus Mse＋CAC were the best combinations and generated polymorphic 655 bands with fragment ranging in size from 50 - 473 bp in size. Populations from central Kenya had the highest levels gene diversity. Most of the variability was partitioned into within populations 71%; P 〈 0.001 implying that collection strategies for conservation should focus on a few populations with many individuals across the ecological amplitude of the population. Genetic differentiation was GST = 0.286, an indication of genetic variation among the populations.