Establishment of Ecotilling for Discovery of DNA Polymorphisms in Brassica rapa Natural Population

Ecotilling is a new approach based on enzyme-mediated heteroduplex cleavage to discover DNA polymorphisms in natural population. We used mung bean nuclease（MBN） instead of routinely used CELI to cleave single base pair mismatches in heteroduplex DNA templates. Nested set of primers were designed to amplify targeted region to avoid the influence of the variation in quality and quantity of the genomic DNA. To reduce the costs in fluorescently labeled primers, we added M13 adapter to 5＇end of gene specific primers to make IRD dye labeled M13 forward and reverse primers possibly universal for different genes. A Brassica rapa ZIP gene homologue was subjected to the analysis to practise the feasibility of the method in polymorphisms detection. Our experiment showed this method is efficient in discovering DNA polymorphisms in Brassica rapa natural population.

DNA Polymorphisms of 5′-Flanking Region of Insulin-Like Growth Factor 1 Gene and Their Association with Reproduction Traits in Goats

Research on the identity of genes and their relationship with traits of economic importance in farm animals could assist in the selection of livestock. In this study, the polymorphisms of insulin-like growth factor 1 （IGF1） gene in 561 goats of ten breeds were detected by polymerase chain reaction （PCR）-single strand conformation polymorphism （SSCP） and their association with litter size and birth weight in three breeds were investigated. The effects of IGF1 polymorphisms on the breeding value for litter size and birth weight were examined using least square methods. Two deletions （CA） were detected in the microsatellite and two mutations （A1637G, T1640C） were found in 5′-flanking regulatory region. No significant association between the polymorphisms in 5′-flanking region of IGF1 and birth weight was found in the three breeds of goats. In Gulin Ma goats, two polymorphisms were found to affect litter size traits. In Chuandong White goats and Guizhou White goats, no significant difference （P0.05） in litter size between goats carrying different genotypes was observed. Further evaluation and confirmation studies in more goat populations with larger sample sizes are necessary.

Analysis of Significance of Unite Examination of AFP and DNA Polymorphism of P3 Promoter of IGF-Ⅱ Gene

The DNA of P3 promoter region of IGF-Ⅱ gene was obtained by means of PCR technique. The examination of DNA polymorphism by restriction endonuclease BstE Ⅱ and the examination of AFP by bioluminescence immunoassay technique were carried out. The results have a significant difference( P <0.005). But the positive rate of AFP is higher than that of DNA polymorphism. The experimental result shows that the change of the DNA polymorphism of IGF-Ⅱis not the only carcinogenic factor. The suggested unite examination is the best method for the diagnosis of the primary hepatocellular carcinoma.

Diversity Suppression-Subtractive Hybridization Array for Profiling Genomic DNA Polymorphisms

Genomlc DNA polymorphlsms are very useful for tracing genetic traits end studying biological diversity among species. Here, we present a method we call the ＂diversity suppresslon-subtractlve hybridization array＂ for effectively profiling genomlc DNA polymorphisms. The method first obtains the subtracted gDNA fragments between any two species by suppression subtraction hybridization （SSH） to establish e subtracted gDNA library, from which diversity SSH arrays are created with the selected subtracted clones. The diversity SSH array hybridizes with the DIG-labeled genomlc DNA of the organism to be assayed. Six closely related Dendrobium species were studied as model samples. Four Dendrobium species as testers were used to perform SSH. A total of 617 subtracted positive clones were obtained from four Dendrobium species, and the average ratio of positive clones was 80.3%. We demonstrated that the average percentage of polymorphlc fragments of palrwlse comparisons of four Dendrobium species was up to 42.4%. A dendrogram of the relatedness of six Dendrobium species was produced according to their polymorphic profiles. The results revealed that the diversity SSH array Is a highly effective platform for profiling genomlc DNA polymorphlsms and dendrograms.

内蒙古地区8种主要蝗虫基因组DNA多态性的RAPD标记研究(英文)

[ Objective] The relationship between the genetic evolution and phylogenesis of the main grasshopper species in Inner Mongolia grasslands in molecular level was studied. [ Method] Random amplified polymorphic DNA （RAPD） technique was used to amplify the 80 individuals of 8 grasshoppers （4 families, 6 genera） in Acridoidea, the polymorphisms of their genomic DNA were compared. [ Result] 64 specific fragments were amplified by 7 primers with the molecular weight of 300 -2 000 bp. The genetic distance between 8 grasshoppers was 0.228 2 -0.589 6. Band pat- tern showed that polymorphism was commonly existed in different genus within the same family and different species within the same genus. The resuits were conducted UPGMA cluster analysis according to Neis＇ genetic distance, the results showed that the species within the same genus first clustered together, then the species in the same family clustered together. [ Condusloa] The study could provide molecular biological basis for system development and evolution research of main grasshoppers in Inner Mongolia grassland.

Heavy metal induced DNA changes in aquatic macrophytes: Random amplified polymorphic DNA analysis and identification of sequence characterized amplified region marker

Plants have been used as good bio-indicators and genetic toxicity of environmental pollution in recent years. In this study, aquatic plants Hydrilla verticillata and Ceratophyllum demersum treated with 10umol/L Cd, 5 umol/L Hg, and 20 umol/L Cu for 96 h, showed changes in chlorophyll, protein content, and in DNA profiles. The changes in DNA profiles included variation in band intensity, presence or absence of certain bands and even appearance of new bands. Genomic template stability test performed for the qualitative measurement of changes in randomly amplified polymorphic DNA （RAPD） profiles, showed significant effect at the given concentration of metals. Cloning and sequencing of bands suggested that these markers although may not be homologous to any known gene but its conversion as a sequence characterized amplified region （SCAR） marker is useful in detecting the effects of genotoxin agents.

外源DNA导入水稻的变异材料随机扩增多态性分析

以小麦、玉米、小米、高粱、狼尾草五个不同属的材料为外源ＤＮＡ供体，经减压渗透转导受体紫稻，从获得的变异材料中各选一份进行了随机扩增多态性ＤＮＡ（ＲＡＰＤ）分析。共用２０个随机引物，其中６个引物扩增出了有差异的多态性ＤＮＡ片段，１个引物可以区分受体材料与变异材料及变异材料间的差异。在以材料两两间的相似率进行的聚类分析中，发现聚类结果与材料变异性状一致，变异材料与受体材料间相似率高达９０．７％，认为变异材料为受体材料在“减压渗透”处理后的真实变异；ＲＡＰＤｓ是检测材料间差异的有效方法。

Apolipoprotein AI gene polymorphisms and risk for coronary artery disease in Chinese Xinjiang Uygur and Han population

Objective To analyze the relationship between polymorphism at the Apolipoprotein AI (Apo AI) gene and the risk for coronary artery disease. Methods A total of 107 patients (mean age 56 ±11 years) diagnosed as having stable angina pectoris (SAP) (23 cases), unstable angina pectoris (UAP) (23 cases) or myocardial infarction (MI) (61 cases) were prospectively evaluated. DNA was obtained from the 107 patients and 50 controls. In order to determine the Apo AI genotypes at two polymorphic sites (G/A at -75 bp, and C/T at+83 bp), DNA was PCR amplified and digested with MspI. Results The frequency of carriers of the rare allele at the - 75 bp site (M1-) was 0.49 in cases and 0.30 in controls (P<0. 05). The frequencies of the M1-allele among patients with SAP, UAP, MI and controls were 0. 37 (vs. controls, P > 0. 05), 0.54 (vs. controls, P < 0.05), 0.52 (vs. controls, P<0. 05) and 0. 30, respectively. The frequencies for carriers of the rare allele at the + 83bp polymorphism (M2) were observed among patients with SAP (0. 09, vs. controls, P > 0.05), UAP (0.11, vs. controls, P>0.05) or MI (0. 12, vs. controls, P>0. 05) and controls (0. 12). There was an slightly increase in the frequency of the Ml - allele in patients with SAP to UAP or MI (0. 37 vs. 0. 54 vs. 0. 52; all P>0. 05) and Ml polymorphism as a risk factor for CAD ( OR = 3. 74, P < 0. 05). In the + 83bp polymorphism there was no difference in the allelelic frequencies in cases and controls (0. 11 vs. 0. 12; P > 0. 05). There was no significantdifference in the frequency of the M2 - allele in patients with SAP to UAP or MI (0.09 vs. 0. 11 vs. 0. 12; all P>0. 05) and M2 polymorphism not as a factor for CAD (OR=0.80, P>0. 05).Plasma lipoprotein values in patients with the allele M1-and M2 - had no different levels than those homozygous for the M1+and M2+(P>0.05). Conclusion Ml polymorphism (M1 - ) may be as a risk factor for CAD and M2 polymorphism (M2 - ) not as a factor for CAD in Chinese Xinjiang Uygur and Han population.

Blast-Resistance Inheritance of Space-Induced Rice Lines and Their Genomic Polymorphism by Microsatellite Markers

To understand the resistance inheritance basis of space-induced rice lines to blast, and to probe mutants＇ genomic DNA polymorphism compared with ground control by microsatellite markers, three space-induced lines were crossed with a highly susceptible variety LTH, and their F1 and F2 populations were inoculated by two representative blast isolates with broad pathogenicity to analyze their resistance inheritance basis. Meanwhile three mutant lines and the ground control were analyzed by 225 rice SSR （simple sequence repeat） primer pairs selected throughout the 12 chromosomes of whole rice genome, to scan the mutagenesis in genome of the mutant lines. The results indicated the blast-resistant genes harbored in these mutant lines were dominant. It was demonstrated that the resistance of mutant H1 to isolate GD0193 and GD3286 was controlled by a single gene, respectively; while mutants H2 and H3 were controlled by two pairs of major genes against isolate GD3286 and H2 showed complicated genetic mechanism to isolate GD0193. H3＇s resistance to isolate GD0193 was verified to be controlled by a single gene. According to the results of SSR analysis, three mutant lines showed different mutant rates as compared with the ground control, and the mutant rates also varied. Resistance genes can be induced from rice by space mutation, and different genomic variations were detected in blast-resistant lines.

Analysis of DNA methylation in different tissues of Fenneropenaeus chinensis from the wild population and Huanghai No. 1

DNA methylation plays an important role in the regulation of gene expression during biological development and tissue differentiation in eukaryotes. A methylation sensitive amplification polymorphism（MSAP） including digestion, pre-selective amplification and selective amplification was optimized to compare the levels of DNA cytosine methylation at CCGG sites in muscle, gill and hemocyte from the wild populations and the selective breeding of Huanghai No. 1 of Fenneropenaeus chinensis, respectively. Significant differences in cytosine methylation levels among three tissues in two populations were detected. The average DNA methylation ratios in muscle, gill and hemocyte of the wild population were 23.1%, 22.3% and 19.7%, while those were 21.4%, 19.6%,and 18.9% in Huanghai No. 1, respectively. The DNA methylation levels of gill from the two populations were highly significant（P〈0.01）, the difference of muscle was significant（P〈0.05）, while in hemocyte, there were no significant differences（P〉0.05）. DNA polymorphic methylation of gill and hemocyte between the wild population and Huanghai No. 1 varies to some extent, while those of muscle kept in a balanced degree. Furthermore,polymorphic methylation was associated with demethylation and methylation of CCGG loci.

DNA methylation patterns of banana leaves in response to Fusarium oxysporum f. sp. cubense tropical race 4

Fusarium wilt of banana, which is caused by Fusarium oxysporum f. sp. cubense tropical race 4 （Foc TR4）, is a serious soil-borne fungal disease. Now, the epigenetic molecular pathogenic basis is elusive. In this study, with methylation-sensitive amplification polymorphism （MSAP） technique, DNA methylation was compared between the leaves inoculated with Foc TR4 and the mock-inoculated leaves at different pathogenic stages. With 25 pairs of primers, 1 144 and 1 255 fragments were amplified from the infected and mock-inoculated leaves, respectively. DNA methylation was both changed and the average methylated CCGG sequences were 34.81 and 29.26% for the infected and the mock-inoculated leaves. And DNA hypermethylation and hypomethylation were induced by pathogen infection during all pathogenic stages. Further, 69 polymorphic fragments were sequenced and 29 of them showed sequence similarity to genes with known functions. And RT-PCR results of four genes indicated that their expression patterns were consistent with their methylation patterns. Our results suggest that DNA methylation plays important roles in pathogenic response to Foc TR4 for banana.

RAPD Markers and Genetic Information Entropy in Environmental Monitoring: A Case Study with Wild Mushrooms

Mushrooms have a remarkable scientific value due to their nutritional, medicinal properties and industrial applications in enzyme production, so that effort in the maintenance of native wild mushroom varieties is increasing. The present study focuses on the use of Random Amplified Polymorphic DNA (RAPD) markers for biodiversity measure of wild mushroom species of the Northwest mountainous region of Greece. Data mining of similarity matrices from RAPD analysis was used to extract measurable entropy parameters for mushroom biodiversity monitoring based on Shannon’s information entropy. Shannon information index provides an easy assessment of the entropy of the genetic information of the germplasm per mushroom species while the total equitability index (EH) = 0.871 offers an overall estimation of the genetic variation evenness of all species in the population of the studied mushrooms. Application of RAPDs with parallel entropy analysis is an easily applicable and low-cost valuable technology in environmental monitoring, using genetic information of wild mushroom species as an indicator that can lead to future actions in biodiversity maintenance and germplasm protection. The provided methodology can serve as a pilot procedure enriched with other environmental factors to monitor and protect wild mushroom communities native to the Greek countryside or in any part of the world and provide comparable results about biodiversity from different regions using common entropy indices.

板栗优良品种(无性系)苗木分子标记鉴别研究

利用RAPD标记对浙江省林木良种审定委员会审定 (认定 )和通过省级鉴定的 8个板栗品种及无性系进行指纹分析 ,鉴别出各板栗品种 (无性系 )。实验过程中对 684个随机寡核苷酸引物进行了重复筛选 ,经筛选共获得 9个稳定的RAPD标记 ,构建了板栗的DNA指纹图谱。经过大量的实验 ,确定了鉴别板栗优良品种 (无性系 )的较为理想的实验程序 ,获得了RAPD良好的重复性 ,建立了较完整的板栗RAPD分析的技术体系 ,用大田苗进行初步验证 。

Molecular Systematic Studies on Chinese Mandarina Silkworm (Bombyx mandarina M.) and Domestic Silkworm (Bombyx mori L.)

Molecular systematic studies on mandarina silkworm (Bombyx mandarina M.) in 11 regions in China and 25 representative strains of domestic silkworm (Bombyx mori L.) were conducted using molecular biology techniques. Results obtained from the analysis of DNA polymorphism and clustering of all the silkworm samples provide new evidence for the view that the domestic silkworm originated from the Chinese mandarina silkworm. On the basis of literature reviewing, a new hypothesis on the origin of the domestic silkworm was put forward. It was thought that the domestic silkworm was most probably domesticated from the Chinese mandarina silkworm of different ecotypes including monovoltinism, bivoltinism and multivoltinism; and that the domestic silkworm had the genetic background of monovoltinism, bivoltinism and multivoltinism at the very beginning of the domestication. The current strains of the domestic silkworm of different voltinism are the evolutionary results of thousands of years of rearing and artificial selections.

Genetic Diversity Caused by Environmental Stress in Natural Populations of Niupidujuan as Revealed by RAPD Technique

Multiplex environmental factors are generally expected to have significant effects on genetic diversity of plant populations.In this study,randomly amplified polymorphic DNA（RAPD） technique was used to reveal the genetic diversity in the same species of four populations collected from Niupidujuan（Rhododendron chrysanthum） at different altitudes,an endangered species,endemic to Northeast China.Initially,twenty informative and reproducible primers were chosen for final RAPD analysis.A total of 152 clear bands were obtained,including 143 polymorphic ones.With the help of POPGENE software,the poly rate was calculated to be 94.07% and the evenness of amplified bands for every primer was 6.8.Additionally,the mean observed number of alleles was 1.7265 with an effective number of 1.3608.An examination of the gene indicated a diversity of 0.2162 with an information diversity index of 0.3313.For these data,the clustering blurred analysis was performed with the aid of NTSYS-pc software to define the Nei＇s gene diversity and the Shannon information diversity index of the four plant populations.The relationships between the genetic diversity indexes on the one hand and the geographic and climatic factors on the other hand were estimated by the Pearson correlation with SPSS 11.0 software.The results of the correlation analysis show that there were significant（P〈0.05） or highly significant（P〈0.01） correlations between each of the genetic diversity indexes and the different temperature which were mainly caused by the altitude different populations located.These data highlight the importance of native populations in shaping the spatial genetic structure in Niupidujuan.

Analysis on Genetic Relationship of Oxya chinensis and Oxya japonica from Xuzhou and Pingshan, China

Genetic relationship among Oxya chinensis populations and Oxya japonica populations collected from Xuzhou City, Jiangsu Province and Pingshan County, Hebei Province, China were analyzed by random amplified polymorphic DNA （RAPD） markers. A total of 125 DNA bands ranging from 200 to 2 200 bp were amplified by 10 random primers in DNA samples from 43 grasshopper individuals. One hundred and twenty-three （99%） of these bands were polymorphic. Shannon＇s index showed that the genetic, diversity within O. chinensis （0.3432） was higher than that of O. japonica （0.2781）. Nei＇s genetic distance between O. chinensis population and O. japonica population from the same area was less than that between populations from two different areas. The dendrogram based on Nei＇s genetic distance of RAPD markers was constructed using the unweighted pair group method with the arithmetic average （UPGMA） and Neighbor-Joining （NJ） methods. Cluster analysis indicated that all the individuals were grouped into two main clusters. O. chinensis populations from Xuzhou and Pingshan formed one cluster, and O. japonica populations from the two regions belonged to another cluster. The results demonstrated that RAPD can detect within species, and among closely related species. polymorphisms to distinguish minor difference among individuals

Rapid detection of self-biting disease of mink by specific sequence-characterized amplified regions

Self-biting disease occurred in most farmed fur animals in the world. The mechanism and rapid detection method of this disease has not been reported. We applied bulked sergeant analysis （BSA） in combination with RAPD method to analyze a molecular genetic marker linked with self-biting trait in mink group. The molecular marker was converted into sequence-characterized amplified regions （SCAR） marker for rapid detection of this disease. A single RAPD marker A8 amplified a specific band of 263bp in self-biting minks, which was designated as SRA8-250, and non-specific band of 315bp in both self-biting and healthy minks. The sequences of the bands exhibited 75% and 88% similarity to Canis familiarizes major histocompatibility complex （MHC） class II region and Macaca mulatta MHC class I region, respectively. A SCAR marker SCAR-A8 was designed for the specific fragment SRA8-250 and validated in 30 self-biting minks and 30 healthy minks. Positive amplification of SCAR-A8 was detected in 24 self-biting minks and 12 healthy minks. χ2 test showed significant difference （p〈0.01） in the detection rate between the two groups. This indicated that SRA8-250 can be used as a positive marker to detect self-biting disease in minks. Furthermore, the finding that self-biting disease links with MHC genes has significant implications for the mechanism of the disease.

RAPD Analysis of Rosa laevigata Michx. from Different Origins

[ Objective] This study aimed to investigate the genetic diversity and phylogenetic relationship of 30 populations of Rosa laevigata Michx. from different origins. [ Method] Using RAPD molecular marker technique, eight effective primers were screened from 35 RAPD random primers for amplification. UPGMA clus- ter analysis was performed using NTSYSpc. ver. 2.02 software. [ Result] A total of 86 loci were amplified using the screened primers, including 84 polymorphic loci, indicating an average polymorphic percentage （PPB） of 97.67%. The similarity coefficients of 30 populations of R. laevigata Michx. ranged from O. 11 to O. 58, and these populations were clustered into groups A, B and C. [ Conclusion ] R. laevigata Michx. exhibits multiple genetic polymorphism. R. laevigata Michx. populations from Guizhou Province have closer genetic relationships.

Genetic diversity of the endangered species Rosa rugosa Thunb.in China and implications for conservation strategies

Rosa rugosa Thunb. is one of the dominant and important shrub species in estuary dunes and shingle beaches of northern China. However, its area of distribution, the number of populations, and the size of each population have decreased rapidly in the past two decades because of habitat degradation and loss. Random amplified polymorphic DNA markers were used to determine the genetic diversity of four remaining large natural populations of R. rugosa and to discuss an effective conservation strategy for this endangered species in China. High genetic variations were detected in R. rugosa populations in China. The mean percentage of polymorphic loci （P%） within four local populations was 57.99%, with the P% of the total population being 75.30%. Mean Shannon＇s information index （H0） was 0.2826, whereas total Ho was 0.3513. The genetic differentiation among populations was 0.1878, which indicates that most genetic diversity occurs within populations. Population Tumenjiang （TMJ） showed the highest genetic diversity （P% = 66.27%; H0 = 0.3117） and contained two exclusive bands. Population Changshandao （CSD） showed higher genetic diversity （P% =59.04%; H0 = 0.3065）. Populations TMJ and CSD contained 95.33% and 99.33%, respectively, of loci with moderate to high frequency （P〉0.05） of the total population. These results indicate that populations TMJ and CSD should be given priority for in situ conservation and regarded as seed or propagule sources for ex situ conservation. The results of the present study also suggest that R. rugosa in China has become endangered as a result of human actions rather than genetic depression of populations; thus, human interference should be absolutely forbidden in R. rugosa habitats.

Genetic diversity and differentiation of pedunculate ( Quercus robur ) and sessile ( Q. petraea ) oaks

This study was conducted to determine the parent-off spring genetic structure of the pedunculate oak(Quercus robur L.),sessile oak(Q.petraea[Matt.]Liebl.)and their hybrids.Forty half-sib Quercus families and their maternal trees originating from one tree stand in southern Lithuania were analyzed using SSR and RAPD markers.Based on a preliminary study of leaf morphological traits,the individuals separated into six groups.The studied halfsib oak families were also compared for allelic diversity,including group variations;genotypic structure;genetic diversity;and the degree of genetic subdivision and diff erentiation.The level of genetic variation and subdivision was lower in the hybrid families than in the families of the parental species.Genotypic analysis of the half-sibling off spring showed the asymmetric nature of interspecifi c hybridization processes of pedunculate and sessile oaks in mixed stands.