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.

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.

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.

Effect of Development Stage on the Artemisinin Content and the Sequence Characterized Amplified Region （SCAR） Marker of High-Artemisinin Yielding Strains of Artemisia annua L.

The effects of development states on the artemisinin content of clone S1 of Artemisia anuua L. grown in a greenhouse were investigated in the present study. The artemisinin content increased gradually during the phase of vegetative growth and reached its highest level at 8-9 mg/g dry weight （DW） when the S1 was 6 months old on a long day （LD） photoperiod. Treatment with 9-18 d of short day （SD） photoperiod resulted in the artemisinin content reaching and being maintained at a higher level （2.059-2.289 mg/g DW）, twofold that of control plants and plants of S1 presented at the pro-flower budding and flower-budding stages. The artemisinin content varied in different parts of the plant. The artemisinin content of leaves was higher than that of florets and branches. The artemisinin content in middle leaves was higher than that of bottom leaves, and then top leaves. Different densities of capitate glands （the storage organ of artemisinin） located on the surface of leaves, florets, and branches explained the variations in artemisinin content in these parts of the plant. The correlation coefficient between artemisinin content and density of capitate glands on the surface of different organs was 0.987. The genetic marker for artemisinin content was screened using random amplified polymorphic DNA （RAPD） and sequence characterized amplified region （SCAR） techniques. The random primer OPAl5 （5＇-TTCCGAACCC-3＇） could amplify a specific band of approximately 1 000 bp that was present in all high-artemisinin yielding strains, but absent in all low-yielding strains in three independent replications. This specific band was cloned and its sequence was analyzed. This RAPD marker was converted into a SCAR marker to obtain a more stable marker.

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。

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.

A Set of SCAR Markers Efficiently Differentiating Hybrid Rice

Molecular markers have been widely used in crop genetic improvement, seed test and genetic mapping. Of which, sequence characterized amplified region （SCAR） markers are particularly popular for its diversity, stable reproducibility, and suitability for analyzing large number of samples. In this study, 500 random amplified polymorphic DNA （RAPD） primers were tested, and a set of SCAR markers comprising 37 pairs of loci-specific primers were developed from the DNA fragments ranging from 300 to 1000 bp which correspond to the stable, distinctive RAPD banding patterns. Using these SCAR markers, 59 hybrid rice combinations were assessed and distinguished into 58 subgroups at the similarity coefficient of 0.97 in a genetic clustering tree based on the allele diversities of the SCAR markers. Furthermore, 13 hybrid rice combinations were reassayed with 40 randomly selected simple sequence repeat （SSR） markers to evaluate the effectiveness of these SCAR markers. SSR markers produced similar results to SCAR markers as the 13 hybrid rice combinations were completely separated at the similarity coefficient of 0.91 in the clustering tree established from SSR patterns. Taken together, SCAR markers prove to be effective tools for identifying and differentiating hybrid rice combinations.

Development and Application of SCAR Markers for Discriminating Cytoplasmic Male Sterile Lines from Their Cognate Maintainer Lines in Indica Rice

The DNAfragments about 1 600 bp were amplified using random amplified polymorphism DNA （RAPD） primer OPAl2 with the templates of mitochondrial DNA of Zhenshan 97A and Zhenshan 97B, and were sequenced. The nucleotide sequences and lengths of the fragments from Zhenshan 97A and Zhenshan 97B showed no difference. The precise length of the fragment was 1 588 bp. Sequence characterized amplification region （SCAR） primers were then developed to discriminate the cytoplasmic male sterile （CMS） lines and their maintainer lines. A specific 1 588 bp fragment could be amplified with SCAR primers, CHI19F2/CHI19R2 and CHI20F3/CHI23R3, in the mitochondrial DNA of Zhenshan 97A, but not Zhenshan 97B. Furthermore, the specific fragment could be also amplified from the total DNA from green leaf tissues of Zhenshan 97A with SCAR primers, but not Zhenshan 97B. With the corresponding primers, the specific fragment could also be amplified from the total DNA of green leaves of other two CMS lines with wild abortive type cytoplasm （CMS-WA）, namely Zhenpin A and Tianfeng A, but not in their maintainer lines. Moreover, using total DNA as template, each of the four pairs of SCAR primers could also be used to amplify the 1 588 bp fragment in CMS-ID （Indonesia paddy type） line 11-32A but not in 11-32B, and the specific fragment was amplified from the DNA of both F1 and F2 seedlings of Shanyou 63. The results of detecting the genetic purity of a man-made mixture of the seeds of Zhenshan 97A using CHI20F3/CHI23R3 were completely consistent with the phenotypes. Taken together, these results indicated that the specific 1 588 bp-fragment amplified by CHI20F3/CHI23R3 was the unique amplification products of CMS mitochondrial DNA, and could be used to distinguish CMS-WA and CMS-ID lines from their corresponding maintainer lines at the seedling stage.

Development of a SCAR marker for male gametophyte of Gracilariopsis lemaneiformis based on AFLP technique

The red alga Gracilariopsis lemaneiformis(Bory) is an economically valuable macroalgae. As a means to identify the sex of immature Gracilariopsis lemaneiformis, the amplifi ed fragment length polymorphism(AFLP) technique was used to search for possible sex- or phase-related markers in male gametophytes, female gametophytes, and tetrasporophytes, respectively. Seven AFLP selective amplifi cation primers were used in this study. The primer combination E-TG/M-CCA detected a specifi c band linked to male gametophytes. The DNA fragment was recovered and a 402-bp fragment was sequenced. However, no DNA sequence match was found in public databases. Sequence characterized amplifi ed region(SCAR) primers were designed from the sequence to test the repeatability of the relationship to the sex, using 69 male gametophytes, 139 female gametophytes, and 47 tetrasporophytes. The test results demonstrate a good linkage and repeatability of the SCAR marker to sex. The SCAR primers developed in this study could reduce the time required for sex identifi cation of Gracilariopsis lemaneiformis by four to six months. This can reduce both the time investment and number of specimens required in breeding experiments.

Characterization and complete genome sequence of vB_EcoP-Bp4,a novel polyvalent N4-like bacteriophage that infects chicken pathogenic Escherichia coli

Pathogenic Escherichia coli cause chicken colibacillosis, which is economically devastating to the poultry in- dustry worldwide （Bagheri et al., 2014）. Owing to in- creasing antibiotic resistance, phage therapy reagents have been developed to treat bacterial infections （Xu et al., 2015）.

Geochemical characterization of loess-paleosol sequences:Comparison between the upper reaches of the Hanjiang and Weihe river valleys,China

This paper aims to compare the geochemical characteristics of loess-paleosol sequences in the upper reaches of the Hanjiang and Weihe river valleyswhich are located in the semi-humid temperate zone and humid subtropical zonerespectively. The Mituosi（MTS） profile in the upper reaches of the Hanjiang River valley and the Yaohecun（YHC） profile in the Weihe River valley were selected for this comparative research. The stratigraphic characteristicscompositionchemical weathering intensityleaching rates of Ca and Namobility of major elementsand transport features of Na and Fe were analyzed with respect to depth and compared between the two profiles. This study reached the following conclusions.（1） The composition of the loess-paleosol sequences in two regions are quite similar to the average composition of the upper continental crust（UCC）indicating that the loess in the two regions came from multiple sources and was mixed well. Thereforethe loess in the two regions is considered aeolian loess.（2） Compared with the loess-paleosol sequence in the Weihe River valleythe loess-paleosol sequence in the upper reaches of the Hanjiang River valley features a darker color; a higher chemical index of alteration（CIA） value; higher leaching rates of Na and Ca; higher migration ratio（relative to K） of AlSiMgand Na; and lower migration ratio of Fe and Ca. This evidence indicates that the loess-paleosol sequence in the humid subtropical environment experienced stronger chemical weathering intensity than the loess-paleosol sequence in the semi-humid temperate zone.（3） Both the YHC profile and MTS profile record a period of climate deterioration at 6000–5000 a BP. The period punctuated the mid-Holocene Climatic Optimum（8500–3100 a BP） in the study area.

An Improved Unsupervised Approach to Word Segmentation

ESA is an unsupervised approach to word segmentation previously proposed by Wang, which is an iterative process consisting of three phases: Evaluation, Selection and Adjustment. In this article, we propose Ex ESA, the extension of ESA. In Ex ESA, the original approach is extended to a 2-pass process and the ratio of different word lengths is introduced as the third type of information combined with cohesion and separation. A maximum strategy is adopted to determine the best segmentation of a character sequence in the phrase of Selection. Besides, in Adjustment, Ex ESA re-evaluates separation information and individual information to overcome the overestimation frequencies. Additionally, a smoothing algorithm is applied to alleviate sparseness. The experiment results show that Ex ESA can further improve the performance and is time-saving by properly utilizing more information from un-annotated corpora. Moreover, the parameters of Ex ESA can be predicted by a set of empirical formulae or combined with the minimum description length principle.

Comparison of Morphological and Genetic Differentiations in Filial Generation of Cross Between Indica and Japonica Rice

A recombinant inbred line （RIL） population of F8 and F9 generations derived from a cross between a typical indica rice （Qishanzhan） and a typical japonica rice （Akihikari） was used to study the difference between morphological differentiation based on phenotype characters and genetic differentiation using indica and japonica specific SSR markers, and to evaluate the relationship between vascular bundle characters and morphological and genetic differentiations. The results showed that the frequency distributions of morphological and genetic differentiations were all inclined to japonica type in the filial generation. The population was more inclined to japonica type based on genetic differentiation than on morphological differentiation. The consistent degrees of classification based on the Cheng’s index, the ratio of large vascular bundle number to small vascular bundle number in panicle neck （RLSVB） and the ratio of large vascular bundle number in the second internode from the top to that in the panicle neck （RLVB） were all about 50% compared with the genetic differentiation, and the consistent degree of the total scores of the Cheng’s index combined with the vascular bundle number ratios was significantly increased to about 80% compared with the genetic differentiation. Therefore, the vascular bundle characters could be used as a helpful supplement for subspecies classification.

Assessment of Genetic Diversities of Selected Laminaria （Laminariales, Phaeophyta） Gametophytes by Inter-Simple Sequence Repeat Analysis

Abstract: Inter-simple sequence repeat (ISSR) analysis was used to assess genetic diversity among 10 pairs of male and female Laminaria gametophytes. A total of 58 amplification loci was obtained from 10 selected ISSR primers, of which 34 revealed polymorphism among the gametophytes. Genetic distances were calculated with the Dice coefficient ranging from 0.006 to 0.223. A dendrogram based on the unweighted pair-group method arithmetic (UPGMA) average showed that most male and female gametophytes of the same species were clustered together and that 10 pairs of gametophytes were divided into four groups. This was generally consistent with the taxonomic categories. The main group consisted of six pairs of gametophytes, which were selected from Laminaria japonica Aresch. by intensive inbreeding through artificial hybridization. One specific marker was cloned, but was not converted successfully into a sequence characterized amplified region (SCAR) marker. Our results demonstrate the feasibility of applying ISSR markers to evaluate Laminaria germplasm diversities.

Molecular authentication of geo-authentic Scrophularia ningpoensis

Scrophularia ningpoensis has long been used in the Chinese Materia Medica for inflammation.Like other herbal medicines,S.ningpoensis collected from different localities may considerably differ in their therapeutic efficacy,and the one grown in Zhejiang Province is recognized as geo-authentic.However,it is difficult to confirm the geo-graphical authenticity by similar morphological characteristics.In the present study,inter-simple sequence repeat(ISSR) markers were conducted to detect S.ningpoensis from different origins.A 1 259-bp fragment amplified by primer UBC874 was found only in geo-authentic ones.By cloning and sequencing that specific band,sequence characterized amplified region(SCAR) markers were designed to distinguish geo-authentic S.ningpoensis from others.This is a rapid and easy method that can be used to identify the geographical authenticity of S.ningpoensis.