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.

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.

APOLIPOPROTEIN E GENE POLYMORPHISMS AND RISK FOR CORONARY ARTERY DISEASE IN CHINESE XINJIANGUYGUR AND HAN POPULATION

Objective To examine the relationship between apolipoprotein E (Apo E) gene polymorphism and risk of coronary artery disease (CAD), analyzing association of polymorphism with classical risk factors. Methods A total of 124 patients (including 84 Han population and 40 Uygur population) with angiographically verified CAD or myocardial infarction were prospectively evaluated. Data referring to hypertension, diabetes, and tobacco consump-tion were recorded. The levels of total cholesterol (TC), high density lipoprotein (HDL) cholesterol, Apo A1 and B, and triglycerides (TG) were determined. DNA was obtained from 124 patients and 70 controls. In order to determine Apo E genotypes, DNA was PCR amplified and digested with HhaI. The genetic polymorphism of Apo E is due to three common alleles, epsilon(ε) 2, ε3, ε4, at a single autosomal gene locus. These alleles determine the six phenotypes E2/2, E3/3, E4/4, E4/2, E4/3, and E3/2. Results In Uygur population, the frequency of the ε2, ε3, and ε4 was 0.155, 0.648, and 0.197 respectively. In Han po-pulation, the frequency of the ε2, ε3, and ε4 was 0.081, 0.772, and 0.146 respectively. In the patient group, the frequency of the ε2, ε3, and ε4was 0.060, 0.758, and 0.182 respectively. In the control group, the frequency of the ε2, ε3, and ε4 was 0.193, 0.671, and 0.136 respectively. ε2 frequency of Uygur’ patients and controls was 0.050 and 0.290 respectively. Serum low density lipoprotein (LDL) cholesterol, TC, and TG values tended to decrease from the Apo E-4 phenotypes to Apo E-2 phenotypes. When deletion polymorphism of ε2 was compared with the common risk factors for CAD, its risk ratio (RR) is 4.38. Conclusions These studies confirm and find that Apo E phenotype distribution in Uygur population differs significantly from that in Han population in Xinjiang. CAD patients have significantly lower ε2 allele and slightly higher ε3 or ε4 allele frequency than controls, especially in Uygur population. It shows protective effects of ε2 on CAD.

Random Amplified Polymorphic DNA (RAPD) Markers on Genomic DNA Polymorphism in 8 Main Grasshoppers in Inner Mongolia

[ 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.

Analysis of genetic diversity for wild and captive green peafowl populations by random amplified polymorphic DNA technique

The genetic diversity of the populations for 14 wild green peafowls (Pavo muticus) and 18 captive green pea-fowls was investigated by using the technology of random amplified polymorphic DNA (RAPD). Totally 161 and 166 ampli-fied bands were obtained by using 23 arbitrary primers to amplify the genomic DNA of wild and captive green peafowls re-spectively. The results showed that the average relative genetic distance of the wild and captive green peafowls popula-tions was 0.0555 and 0.1355, respectively, and difference of the average relative genetic distances between the two popu-lations was 0.1635. The Shannon diversity index for the wild and captive green peafowl populations was 0.4348 and 1.0163, respectively, which means that there exists significant difference in genetic diversity between the two populations, and the genetic diversity of wild green peafowl was low. The two populations originated from two different families according to analysis by the UPGMA method. This research can provide the theoretical basis for supervising genealogies management of peafowl populations.

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.

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.

Combined Effects of Cadmium and Butachlor on Microbial Activities and Community DNA in a Paddy Soil

Due to frequent soil Cd contamination and wide use of butachlor in China,there is a need to assess their combined toxicity to soil microorganisms.The combined effects of cadmium(Cd,10 mg kg-1 soil) and herbicide butachlor(10,50,and 100 mg kg-1 soil) on enzyme activities and microbial community structure in a paddy soil were assessed using the traditional enzyme assays and random amplified polymorphic DNA(RAPD) analysis.The results showed that urease and phosphatase activities were significantly reduced by high butachlor concentration(100 mg kg-1 soil).When the concentrations of Cd and butachlor added were at a ratio of 1:10,urease and phosphatase activities were significantly decreased whereas enzyme activities were greatly improved at the ratio of 1:5,which indicated that the combined effects of Cd and butachlor on soil urease and phosphatase activities depended largely on their addition concentration ratios.Random amplified polymorphic DNA(RAPD) analysis showed loss of original bands and appearance of new bands when compared with the control soil.Random amplified polymorphic DNA fingerprints suggested substantial differences between the control and treated soil samples,with apparent changes in the number and size of amplified DNA fragments.The addition of high concentration butachlor and the combined impacts of Cd and butachlor significantly affected the diversity of the microbial community.RAPD analysis in conjunction with other biomarkers such as soil enzyme parameters would prove a powerful ecotoxicological tool.Further investigations should be carried out to understand the clear link between RAPD patterns and enzyme activity.

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.

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.

Use of random amplified polymorphic DNA （RAPD） analysis to detect jujube witches＇ broom phytoplasmas

Jujube witches＇ broom is a devastating disease of Ziziphusjujube that occurs in various jujube regions of China. Nucleic acid extracted from midribs of samples collected from three jujube varieties （＂Suanzao＂, ＂Lajiaozao＂ and ＂Langzao＂） from symptomatic and asymptomatic shoots were tested by random amplified polymorphic DNA analyses. Using 13 different 10 and 11-bp random primers the amplification of jujube DNA was achieved from all the samples; AMI4 primer provided amplification of specific DNA fragments of about 400 bp, only from samples collected from symptomatic plants. No genetic variations in these varieties were identified using the other 11 arbitrary primers; only with primer AL07 it was possible to differentiate ＂Langzao＂ from the other two varieties tested. All the experiments were repeated 2 times and the results were consistent. Compared with PCR analyses with phytoplasma-specific primers, RAPD techniques resulted to be an alternative rapid and sensitive method for detecting jujube phytoplasmas presence in different jujube varieties.

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.

Intergeneric Somatic Hybrid Plants Between Citrus and Poncirus trifoliata and Evaluation of Their Root Rot Resistance

Protoplasts of Page tangelo (Citrus reticulata Blanco×C. paradisi Macf.) cell suspension culture were electrically fused with mesophyll protoplasts isolated from trifoliate orange (Poncirus trifoliata (L.) Raf.). More than 150 plantlets regenerated after 4-5 months of culture. The regenerated plants were trifoliate with well developed root systems. Root_tip chromosome counting of more than 20 randomly selected plants revealed that they were all tetraploids (2n=4x=36). RAPD analysis of 7 randomly selected plants verified their hybridity. Inoculation of citrus Phytophthora parasitica Dastar toxin on leaves of somatic hybrids and both parental genotypes showed that Page tangelo was moderately susceptible, and trifoliate orange was highly resistant while the somatic hybrids were resistant. The potential of this somatic hybrid as rootstock is also discussed.

Cytological and Molecular Identification of Alien Chromatin in Giant Spike Wheat Germplasm

Alien chromosomes of twelve giant spike wheat germplasm lines were identified by C-banding, genomic in situ hybridization (GISH), sequence characterized amplified region (SCAR), and random amplified polymorphic DNA (RAPD). All lines showed a chromosome number of 2n = 42, five of them carried both a pair of wheat-rye (Triticum aestivum-Secale cereal) 1BL/1RS translocation chromosomes and a pair of Agropyron intermedium (Ai) chromosomes, three carried a pair of Ai chromosomes only, three others carried a pair of 1BL/1RS chromosomes only, and one carried neither 1BL/1BS nor Ai chromosome. Further identification revealed that the identical Ai chromosome in these germplasm lines substituted the chromosome 2D of common wheat (T aestivum L.), designated as 2Ai. The genetic implication and further utilization of 2Ai in wheat improvement were also discussed.

Analysis and identification of SCAR molecular markers associated with birch fiber length trait

The fiber length trait （FLT） of 538 individuals from nature birch population in Maorshan region, Heilongjang, China were measured, of which 100 individuals were selected as representative variety of correlated fragments screening with random amplified polymorphism DNA （RAPD） technique. In total of 20 RAPD primers were tested through multiple regression analysis between amplified strip and the character behaviors, and a correlative segment BFLR-16 was obtained. The correlation coefficient between BFLI-16 and FLT was 0.6144, with the significant level of 1%. BFLI-16 was then cloned, sequenced and transformed into SCAR marker. The percentage of identifying long fiber birches by this SCAR was more than 92. The result indicates that the SCAR markers has high specificity for the long fiber individuals and is highly linked with the gene controlling the character of fiber length, and its existence is significantly correlative with the increase in the fiber length.

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.

RAPD analysis of natural populations of Acanthopanax brachypus

Random Amplified Polymorphic DNA (RAPD) analysis is a new technology of molecular marking which has proved very powerful in detecting genetic diversity at the level of population. The genomic DNAs used in our experiment were extracted from fresh leaves taken from 59 individuals sampled from three natural populations in Yan An, Shanxi Province. Through more than 2,000 PCRs, deep-going RAPD analysis was carried out on DNA samples from 49 inviduals. The percentage of polymorphic RAPD loci found in these three populations were respectively 27.2 %, 18.6 % and 5.4 %; the average genetic distances within population, 0.055, 0.036 and 0.008; the average genetic distances between populations (Ⅰ-Ⅱ), (Ⅰ-Ⅲ) and (Ⅱ-Ⅲ), 0.105, 0.096 and 0.060. The genetic diversity of A. brachypus within and between populations was found, for the first time, to be rather poor,thus revealing innate factors as the cause contributing to its endangered status. In addition, our work also provides basic materials for elucidating the underlying cause of its endangerment and for its protection biology.

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.