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.

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.

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.

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.

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.

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.

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。

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。

江浙地区菱品种遗传多样性的SLAF-seq分析

利用SLAF-seq技术对江浙地区17个主要的栽培菱品种和1个野生菱种质进行高通量测序,获得445594个SLAF标签,鉴定到多态性SLAF标签95931个。通过序列分析,获得269338个SNP标记,并基于这些SNP构建18份菱样品的遗传发育树及进行群体结构分析。结果表明,SLAF-seq技术能高效地开发出大量可用于群体遗传分析的SNP标记,基于SNP标记构建的进化树能较好地区分不同类型的菱品种。该结果对开展菱种质资源鉴定和菱种质遗传演化研究具有重要参考价值。

山桐子性别分子标记的通用性验证

采用已知性别的120份山桐子(Idesia polycarpa Maxim.)样品,检验了2个已报道UBC841和STZ分子标记在山桐子性别鉴定中的通用性。同时,分析了大量简单重复序列间区扩增多态性(ISSR)和相关序列扩增多态性(SRAP)标记在山桐子雌雄株中的多态性。结果表明,UBC841标记在24份山桐子样品中可检测到多态性条带,但未检测到雌雄性别特异性条带;STZ标记可在部分样本中扩增出目的条带,但未表现出雌雄株性别特异性;说明这2组标记均未表现出良好的通用性,雌雄性别鉴别准确率较低。此外,筛选了26条ISSR引物和780对SRAP引物组,也未能获得雌雄性别特异标记,说明采用通用型分子标记ISSR和SRAP在山桐子中较难筛选到稳定、可靠的性别特异的标记。

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。

Analysis of the Genetic Structure of Sclerotinia sclerotiorum （Lib.） de Bary Populations from Different Regions and Host Plants by Random Amplified Polymorphic DNA Markers

The genetic diversity and genetic structure of a population of isolates of Sclerotinia sclerotiorum (Lib.) de Bary from different regions and host plants were investigated using the random amplified polymorphic DNA (RAPD) method with 20 random decamer primer pairs in order to provide some information on the phylogenetic taxa and breeding for resistance to sclerotinia stem rot. A minimum of three and a maximum of 15 unambiguously amplified bands were generated, furnishing a total of 170 bands ranging in size from 100 to 3 200 bp, corresponding to an average of 8.5 bands per primer pair. One hundred and four of these 170 bands (61.2%) were polymorphic, the percentage of polymorphic bands for each primer pair ranging from 0.0% to 86.7%. The genetic relationships among the isolates, based on the results of RAPD analysis, were examined. The genetic similarity of all selected isolates was quite high. At the species level, the genetic diversity estimated by Nei's gene diversity (h) was 0.197 and Shannon's index of diversity (I) was 0.300. The unweighted pair-group mean analysis (UPGMA) cluster analysis showed that most isolates from the same regions were grouped in the same cluster or a close cluster. The population of isolates from Hefei (Anhui Province, China) was more uniform and relatively distant to other populations. The Canadian population collected from carrot (Daucus carota var. sativa DC.) was relatively close to the Polish population collected from oilseed rape (Brassica napus L.) plants. There was no relationship between isolates from the same host plants. An analysis of molecular variance (AMOVA) revealed that the percentage of variance attributable to variation among and within populations was 50.62% and 49.38%, respectively. When accessions from China, Europe, and Canada were treated as three separate groups, the variance components among groups, among populations within groups, and within populations were ?0.96%, 51.48%, and 49.47%, respectively. The genetic differentiations among and within populations were highly significant (P < 0.001). Similarly, the coefficient of gene differentiation (Gst) in total populations calculated by population genetic analysis was 0.229 4, which indicated that the genetic variation among populations was 22.94%. The gene flow (Nm) was 1.68, which indicated that the gene permutation and interaction among populations was relatively high.

扁蓿豆种内杂种鉴定及其F1和F2代主要农艺性状优势分析

扁蓿豆是一种抗逆性强,遗传多样性丰富的豆科牧草,可作为同属牧草的杂交育种材料,为其提供丰富的基因资源。而如何获得真杂交种是开展杂交育种和杂种优势利用的重要基础。为获得遗传性状更加丰富的扁蓿豆新种质材料,以株型直立且高产的直立型扁蓿豆和抗逆性强的野生扁蓿豆(黄花变异材料,简称黄花扁蓿豆)为亲本,采取正反交配置了两个杂交组合。通过相关序列扩增多态性(SRAP)分子标记技术从118个杂交F1代中鉴定出85个真杂种。从直立型扁蓿豆♀×黄花扁蓿豆♂的杂种F1代中获得其F2代,进一步对表现优良的13株F1代和20株F2代的株高、株型指数、一级分枝数、近似叶面积、叶茎比和单株地上生物量等主要农艺性状进行杂种优势分析。结果表明:F1代各性状的表型值变异系数为18.82%~45.72%,其中单株地上生物量、叶茎比、一级分枝数变异较大。各性状中亲优势为-54.2%~26.9%,其中叶茎比(8.22%)表现出超亲优势。F2代各性状变异系数(19.66%~41.63%)较F1代减小,除近似叶面积(12.69%)、株型指数(17.46%)和叶茎比(35.38%)外其余性状中亲优势(-52.38%~-19.75%)均减弱。F1与F2代产量相关性状的中亲优势和超亲优势多为负向,种内杂种优势较弱。研究结果将为扁蓿豆及苜蓿属其余牧草的品种改良提供新种质。

航天诱变黔草1号高羊茅分子多样性研究

试验以黔草1号为材料,采用RAPD分子标记技术对经航天搭载后的黔草1号高羊茅子代进行遗传多样性分析,探讨空间环境对其遗传多样性的影响。结果表明,利用RAPD引物进行PCR扩增,产生的扩增条带中,多态性条带占83.85%;34份供试材料的相似系数在0.5364~0.7411之间,说明航天搭载黔草1号高羊茅可以造成其遗传物质的变化,诱变后的各株系发生了不同程度的遗传变异,可以从航天诱变后的株系中选择符合育种目标的材料。RAPD聚类分析将供试材料分为七大类。总体来看,具有相似性状的株系基本上能聚在一类,呈现出一定的性状差异。