Optimization of a Reaction System of Sequence Related Amplified Polymorphism and Segregation of Polymorphic Loci in an F_2 Population of Rice

An effective PCR protocol for detecting the sequence related amplified polymorphism （SRAP） in rice was developed. One hundred and ten pairs of SRAP primers were used for segregation analysis in an F2 population derived from a cross between Shennong 606 and Lijiangxintuanheigu. Among the 110 primer pairs, 35 pairs generated 143 polymorphic bands with an average of 4.09 polymorphic bands per primer pair, and 24 pairs （16.78%） showed the genetic distortion （P〈0.05）. Of the 24 primer pairs, 12 pairs deviated toward the male parent Shennong 606 and 11 pairs toward the female parent Lijiangxintuanheigu, only one toward heterozygote. It was found that the segregation distortion might be caused by the joint gametic and zygotic effects.

Application of restriction site amplified polymorphism (RSAP) to genetic diversity in Saccharina japonica

Restriction site amplified polymorphism (RSAP) was used, for the first time, to analyze the genetic structure and diversity of four, mainly cultivated, varieties of the brown alga, Saccharina japonica. Eighty-eight samples from varieties "Rongfu", "Fujian", "Ailunwan" and "Shengchanzhong" were used for the genetic analyses. One hundred and ninety-eight bands were obtained using eight combinations of primers. One hundred and ninety-one (96.46%) were polymorphic bands. Nei's genetic diversity was 0.360, and the coefficient of genetic differentiation was 0.357. No inbreeding-type recession was found in the four brown alga varieties and the results of the "Ailunwan" variety using samples from 2 years showed that the variety was becoming less diverse during the selection inherent in the breeding program. Genetic diversity and cluster analyses results were consistent with these genetic relationships. The results show the RSAP method is suitable for genetic analysis. Continuous inbreeding and selection could reduce the genetic diversity effectively; therefore periodical supervision is required.

Genetic Variation in <i>Salamandra</i><i>infraimmaculata</i>from Different Habitats Using Amplified Fragment Length Polymorphism

The purpose of the present study was to examine the genetic variation in Salamandra infraimmaculata from different breeding site habitats using the Amplified Fragment Length Polymorphism (AFLP) method. The results of the dendogram from a hierarchical cluster analysis show that the grouping of S. infraimmaculata as cluster 5 differs from all the other clusters, including the St1 (Tel-Dan stream) population, which was the most predictable. Five Haplogroups (Hg) were characterized. The mean number of alleles per locus in each population (Ne) ranged from 10.566 (Sp1) to 2.720 (Po6). An average estimated heterozygosity (He) by population ranged from 0.100 (Po6) to 0.186 (St1). Population St1, a permanent breeding site where water was available all year round, exhibited the highest level of polymorphism, while population Po6, from the ephemeral breeding site, exhibited the lowest level of polymorphism. Gene flow between clusters showed that clusters 3 and 4 are sources of migrants and also receive gene flow, while clusters 1 and 2 may be a source of migrants but may not receive much gene flow. A phylogenetic analysis, based on clustering using Nei’s genetic distance, demonstrated that the Tel-Dan population is located on a separate branch within its sub-population. The conclusion of the present study shows that the genetic divergence among isolated populations is not correlated to distance but is affected by the variation of habitats.

A Polymerase Chain Reaction Based DNA Fingerprinting Technique: Amplified Fragment Length Polymorphism for Molecular Typing of Perchlorate Reducing Bacteria

Genetic profiling of environmentally important organisms is very essential for easy identification of biodegrading bacteria. In the previous study, we have reported the perchlorate biodegrading bacteria and characterized them by biochemical analysis and 16 S sequencing. We have observed a very similar isolates of Arthrobacter (Actinobacteria) degrading 4.1 mM and 4.7 mM of ammonium perchlorate [1] 08D0C9EA79F9BACE118C8200AA004BA90B02000000080000000E0000005F005200650066003300390038003100390037003300340037000000 . In this study, we report PCR based DNA fingerprinting technique to generate the genomic signature of these closely related group of Arthrobacter species. This study also effectively generates unique genomic signature for each of these isolates that has potential for use in molecular monitoring as well as for tracking genomic variation and rearrangements.

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.

A Cleaved Amplified Polymorphic Sequence Marker to Detect Variation in Wx Locus Conditioning Translucent Endosperm in Rice

The translucent endosperm trait in a japonica rice variety ＇Kantou 194＇ is controlled by a Wx-mq gene which is allelic to Wx locus by genetic analysis and allelic test. The amylose content analysis showed that an intermediate amylose content between those of glutinous and non-glutinous rice existed in endosperm of homozygous Wx-mq genotype. The slight changes of amylose content in different varieties and F1 grains with an identical Wx-mq genotype might be influenced by dissimilar genetic background. To identify the Wx-mq genotype simply and rapidly, a cleaved amplified polymorphic sequence （CAPS） marker was designed. The result from the molecular detection indicated that it could be used for marker-assisted selection for low amylose content varieties in rice breeding.

Authentication and Genetic Origin of Medicinal <i>Angelica acutiloba</i>Using Random Amplified Polymorphic DNA Analysis

Some Angelica species are used for medicinal purposes. In particular, the roots of Angelica acutiloba var. acutiloba and A. acutiloba var. sugiyamae, known as “Toki” and “Hokkai Toki”, respectively, are used as important medicinal materials in traditional Japanese medicine. However, since these varieties have recently outcrossed with each other, it is difficult to determine whether the Japanese Angelica Root material used as a crude drug is the “pure” variety. In this study, we developed an efficient method to authenticate A. acutiloba var. acutiloba and A. acutiloba var. sugiyamae from each other and from other Angelica species/varieties. The random amplified polymorphic DNA (RAPD) method efficiently discriminated each Angelica variety. A. acutiloba var. sugiyamae was identified via a characteristic fragment amplified by the decamer primer OPD-15. This fragment showed polymorphisms among Angelica species/varieties. The unique fragment derived from A. acutiloba var. sugiyamae was also found in one strain of A. acutiloba var. acutiloba, implying that this strain arose from outcrossing between A. acutiloba var. acutiloba and A. acutiloba var. sugiyamae. This RAPD marker technique will be useful for practical and accurate authentication among A. acutiloba var. acutiloba, A. acutiloba var. sugiyamae, and their adulterants.

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.

Genetic Polymorphism of Wx Gene and Its Correlation with Main Grain Quality Characteristics in Rice

The allelic variation of the Wx gene in 50 non-glutinous rice varieties （lines） was analyzed by using the microsatellite marker RM190 [for （CT）n simple sequence repeat （SSR）] and cleaved amplified polymorphic sequence（CAPS） marker 484/W2R-ACCⅠ[for G/T single nucleotide polymorphism （SNP）]. Six homozygous （CT）n types, namely （CT）20, （CT）19, （CT）18, （CT）17, （CT）16, （CT）14, （CT）11 and （CT）10, and a heterozygous genotype （CT）11/（CT）18 were detected for RM190, of which （CT）11 and （CT）18 were predominant. Two homozygous Wx genotypes （G/G and T/T） and one heterozygous （G/T） were detected using 484/W2R-ACC Ⅰ. Most of the materials with a RM190 of （CT）11 were G/G for SNP of 484/W2R-ACC Ⅰ, while T/T for SNP was predominantly appeared in materials with （CT）18. The materials tested could be grouped into 10 categories using the two markers together. Results indicated that 59.3% variance of amylose content was attributed to the polymorphism of Wx gene revealed by RM190, while 56.1% and 24.6% of the variances in amylose content and gel consistency were respectively to the polymorphism of Wx gene revealed by 484/W2R-ACC Ⅰ. Furthermore, with both SSR and CAPS markers, 72.4% of the variance in amylose content could be explained. In addition, the application prospects of the two markers in breeding were also discussed.

Development of Sequence Characterized Amplified Region (SCAR) Primers for the Detection of Resistance to Sporisorium reiliana in Maize

Head smut of maize （Zea mays L.）, which was caused by Sporisorium reiliana, occurred in most of the maize growing areas of the world. The purpose of this study was to develop SCAR markers for map-based cloning of resistance genes and MAS. Two sets of BC3 progenies, one （BC3Q） derived from the cross Qi319 （resistance）×Huangzao 4 （susceptible）, the other （BC3M） from Mol7 （resistance）× Huangzao 4 （susceptible）, were generated. Huangzao 4 was the recurrent parent in both progenies. A combination of BSA （bulked segregant analysis） with AFLP （amplified fragment length polymorphism） method was applied to map the genes involving the resistance to S. reiliana, and corresponding resistant and susceptible bulks and their parental lines were used for screening polymorphic AFLP primer pairs. One fragment of PI3M61-152 was converted into SCAR （sequence charactered amplified fragment） marker S130. The marker was mapped at chromosome bin 2.09, the interval of a major QTL region previously reported to contribute to S. reiliana resistance. Furthermore, S130 was highly and facilitate map-based cloni associated with resistance to S. reiliana, and could be useful for marker-assisted selection ng of resistance genes.

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.

Panax ginseng-specific sequence characterized amplified region (SCAR) marker for testing medicinal products

To screen genetic polymorphisms of Panax ginseng, as well as those of Panax quinquefolium and Panax notoginseng, analysis of random amplified polymorphic DNA （RAPD） was performed using 120 random primers. Of the successful amplicons obtained, the Panax ginseng-specific RAPD marker C-12 was cloned into a TA vector and sequenced （Genl3ank access number KU553472）. Based on the sequence analysis results, a pair of primers specific to C-12 was designed. Finally, a SCAR marker-based identification system for Panax ginseng was developed after optimization of the reaction conditions. Using this method, two positive bands were stably observed at 300 bp and 130 bp in 33 batches of Panax ginseng samples tested, while negative results were obtained for another 101 batches of samples, including Panax quinquefolium, Panax notoginseng, adulterants, and other medicinal herbs. Thus, we successfully developed a PCR-based method for rapid and effective identification of Panax ginseng, which can be effectively used for the protection and utilization of germplasm resources and identification of the origins of Panax ginseng samples.

基于SCAR标记和DNA条形码技术的苍术基原鉴别研究

目的开发出能同时鉴别北苍术和关苍术的分子标记方法,并探究不同种质资源苍术的遗传进化关系。方法对不同地区北苍术Atractylodes chinensis(Bunge)Koidz及关苍术A.japonica Koidz.ex Kitam基因组DNA的差异片段进行测序,结合SRAP、ISSR、DAMD分子标记方法,优化PCR反应体系,筛选并转换成特异性标记,同时,采用条形码方法分析种间序列差异。结果通过SRAP、ISSR、DAMD三种分子标记方法的PCR扩增,共筛选出198对能稳定扩增且重现性好的引物,转换出7对能稳定、快速鉴别北苍术和关苍术的SCAR引物。条形码方法检测出北苍术ITS2序列长度为454 bp,关苍术ITS2序列长度为453 bp,与其他苍术属植物之间遗传距离较远。NJ树结果显示,北苍术、关苍术及其他苍术属植物均各自聚为一支,表现出良好的单系性。依据ITS2二级结构,4种苍术属植物在螺旋区的茎环数目、大小、位置均有明显差异,可以直观地进行区分。结论所开发的特异性SCAR标记为苍术属植物优良品种的筛选提供了新方法,DNA条形码能稳定、准确鉴别北苍术。

黑果枸杞MSAP技术体系的构建

为利用甲基化敏感多态性扩增技术(MSAP)构建黑果枸杞MSAP技术反应体系,本试验采用CTAB法提取黑果枸杞DNA,通过双酶切、预扩增和选择性扩增对16对引物的多态性进行初步筛选并构建MSAP技术体系。结果表明:双酶切进行12 h,DNA模板能够酶切完全;预扩增反应产物条带集中在250~1000 bp处,选择性扩增可筛选出6对引物组合,条带明显。本研究为后续黑果枸杞DNA甲基化水平的相关分析研究奠定了基础。

基于RAPD技术对大曲芽孢杆菌近似菌株的分类研究

大曲是浓香型白酒酿制过程中最主要的微生物源,大曲中微生物的类型、丰度、新陈代谢活动等均会对浓香型白酒品质产生很大的影响。为了探究大曲中芽孢杆菌的分类及多样性,利用菌株随机扩增多态性DNA标记(random amplified polymorphic DNA,RAPD)分型和16S rDNA序列分析技术,对已分离出的芽孢杆菌进行RAPD分型和16S r DNA序列分析。结果表明,37株芽孢杆菌中有22株枯草芽孢杆菌、9株蜡样芽孢杆菌、3株地衣芽孢杆菌,其中枯草芽孢杆菌又可细分为8类,蜡样芽孢杆菌可细分为3类,地衣芽孢杆菌可分为3类;RAPD分型可以为大曲中芽孢杆菌的快速分型及后续的分类学研究提供理论依据,为研究芽孢杆菌在白酒酿造中的作用奠定基础。

Genetic Mapping of Laminaria japonica and L. Iongissima Using Amplified Fragment Length Polymorphism Markers in a ＂Two-Way Pseudo- Testcross＂ Strategy

With a ＂two-way pseudo-testcross＂ mapping strategy, we applied the amplified fragment length polymorphism （AFLP） markers to construct two moderate density genetic linkage maps for Laminaria. The linkage maps were generated from the 60 progenies of the F1 cross family （Laminaria iongissima Aresch. × L. Japonica Miyabe） with twenty pairs of primer combinations. Of the 333 polymorphic loci scored in 60 progenies, 173 segregated in a 1：1 ratio, corresponding to DNA polymorphisms heterozygous in a single parent, and the other 58 loci existing in both parents followed a 3：1 Mendelian segregation ratio. Among the loci with 1：1 segregating ratios, 79 loci were ordered in 14 linkage groups （648.6 cM） of the paternal map, and 72 loci were ordered in 14 linkage groups （601.9 cM） of the maternal map. The average density of loci was approximately 1 per 8 cM. To Investigate the homologies between two parental maps, we used 58 loci segregated 3：1 for further analysis, and deduced one homologous linkage group. The linkage data developed in these maps will be useful for detecting loci-controlling commercially important traits for Laminaria.

Analysis of Genetic Diversity and Relationships in a Tunisian Fig （Ficus carica） Germplasm Collection by Random Amplified Microsatellite Polymorphisms

The random amplified mirosatellite polymorphism method was performed in a set of Tunisian fig landraces using eighteen primer combinations. A total of sixty three random amplified microsatellite polymorphism （RAMPO） markers were scored and used either to assess the genetic diversity in these cultivars or to detect cases of mislabeling. Opportunely, data proved that the designed procedure constitutes an attractive and fast method with low costs and prevents radio exposure. As a result, we have identified the primer combinations that are the most efficient to detect genetic polymorphism in this crop. Therefore, the derived unweighted pair-group method with arithmetic averages （UPGMA） dendrogram illustrates the genetic divergence among the landraces studied and exhibits a typically continuous variation. Moreover, no evident correlation between the sexes of trees was observed. In addition, using these markers, discrimination between landraces has been achieved. Thus, random amplified mirosatellite polymor- phism is proved to be powerful for characterizing the local fig germplasm.

基于AFLP分子标记的鸭绿江茴鱼遗传多样性分析

为探讨鸭绿江茴鱼群体遗传结构及分化水平,为其种质资源保护提供遗传学依据,利用扩增片段长度多态性(AFLP)方法,对分别采自吉林省内鸭绿江上游、松花江上游和临江金鲨养殖场3个群体的总计90尾鸭绿江茴鱼的遗传多样性进行了比较研究。结果显示,鸭绿江上游、松花江上游和临江金鲨养殖场3个群体的观测等位基因数(Na)分别为1.340 9、1.251 7和1.269 1,有效等位基因数(Ne)分别为1.153 8、1.130 0和1.144 1,Nei’s遗传多样性指数分别为0.093 2、0.078 2和0.086 3,Shannon’s信息指数分别为0.144 7、0.119 8和0.131 9,遗传分化系数(G_(st))为0.176 7~0.254 7,平均值为0.219 4,遗传相似性为0.820 6~0.936 3。UPGMA聚类分析结果显示,松花江上游群体单独聚为一支,鸭绿江上游群体和临江金鲨养殖场群体出现了聚群情况。结果表明,采样的3个鸭绿江茴鱼群体均具有较高的遗传多样性水平,3个群体发生了显著的遗传分化。

Genetic instability in cancer tissues analyzed by random amplified polymorphic DNA PCR

OBJECTIVE: To detect DNA and chromosomes instabilities during the progression of tumors and screen new molecular markers coupled to putative or unknown oncogenes and/or tumor suppressor genes. METHODS: Five kinds of tumors, in a total of 128 specimens, were analyzed by random amplified polymorphic DNA (RAPD) PCR. Bands representing instabilities were recovered, purified, and cloned, labeled as probes for Southern and Northern blot analysis and DNA sequencing. RESULTS: Sample 5 and 3 of the gastric cancer tissues showed the highest genomic changes and the average detectability in five cancers was up to at least 40% (42.2% - 49.4%). There were significant differences in the ability of each primer to detect genomic instability, which ranged from 27% (primer 8) to 68% (primer 2). Band B is a single copy fragment, and was found to be an allelic loss in gastric and colon cancers. It is a novel sequence and was registered in GenBank with Accession Number AF151005. Further analysis revealed that it might be part of a cis-regulatory element of a new tumor suppressor gene, containing a promoter of cis-action 'CACA' box, an enhancer of 'GATA' family and a start codon. CONCLUSIONS: It was impossible or difficult to get great achievements for cancer treatments with the procedure of gene therapy only to one oncogene or one tumor suppressor gene because the extensive DNA variations occurred during the progression of tumor. RAPD assay connected with other techniques was a good tool for the detection of genomic instabilities and direct screening of some new molecular markers related to tumor suppressor genes or oncogenes.

随机扩增多态性DNA分子标记技术在肠球菌分型中的应用

该研究旨在探索随机扩增多态性DNA(Random Amplified Polymorphic DNA,RAPD)分子标记技术在肠球菌种间快速分型的效果。试验通过筛选模板、单引物、双引物及RAPD反应体系,构建肠球菌种间分型体系,对32株已知肠球菌分型确定分型相似度标准,并通过对46株未知肠球菌进行种间分型以及管家基因rpoA测定验证分型效果。结果显示微波法提取基因组DNA满足实验需要,筛选出7条单引物用于双引物配对组合,最适的双引物反应体系为:2×PCR Taq Mix 12.5μL,模板DNA 1μL,引物各1μL,ddH_(2)O 9.5μL;分型效果最好的双引物组合为Primer1+Primer5,该引物组合可以在80%的相似度下区分肠球菌种间差异,Primer1+Primer3组合在500 bp位置扩增出海氏肠球菌的特征条带,可以作为海氏肠球菌的鉴定指标,用以快速鉴定海氏肠球菌。