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.

STUDY ON THE SEQUENCE STRUCTURE OF SBR BY ^(13)C-NMR METHOD Ⅱ. PEAK ASSIGNMENT FOR ALIPHATIC CARBONS SPECTRA

The study on ^(13)C-NMR spectra of aliphatic carbon region of emuision-processed and solution-processed (by lithium catalyst) SBR was carried out. The assignments for more than thirty odd peaks observed experimentally were made by using 'corresponding analysis' method, combined with the empirical parameters reported in literature. The peak intensifies were calculated based on BemouUian statistic assumption.

The Genetic Structure and Diversity of Repomucenus curvicornis Inhabiting Liaoning Coast Based on Mitochondrial COⅠ Gene and Control Region

[Object] This study was conducted to explore the genetic diversity and structure of the wild Repomucenus curvicornis inhabiting Liaoning Coast, China. [Method] The mitochondrial COⅠ gene and control region（CR） were PCR amplified from the wild R. curvicornis populations from the Liaodong Bay（n=22） and the northern Yellow Sea（n=18）, sequenced and analyzed for genetic diversity. [Result] The contents of A, T, C and G of 624 bp COⅠ gene were 24.09%, 31.04%, 25.28%, and 19.59%, and those of 460 bp CR fragment were 32.96%, 32.80%, 14.86% and 19.38%, respectively. The total number of variable sites, average number of nucleotide differences（ k）, haplotype diversity（H） and nucleotide diversity（π） based on COⅠ gene were 38, 4.67,（0.96±0.02） and（0.007 5±0.004 2）, and those based on CR fragment were 26, 3.35,（0.97 ±0.02） and（0.007 3±0.004 3）, respectively. Based on mitochondrial COⅠ gene and CR, the genetic diversity of Liaodong Bay population was lower than that of the northern Yellow Sea population. The AMOVA analysis based on CR fragments revealed almost significant genetic divergence between the Liaodong Bay and the northern Yellow Sea populations, while there was no significant genetic divergence based on COⅠ gene. The results showed that CR and COⅠ gene are effective molecular markers for detecting the genetic diversity of R. curvicornis population, while CR is more reliable than COⅠ gene in detecting the genetic structure. [Conclusion] CR is an appropriate marker for genetic analysis of marine fish population.

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。

Impact of Different Rates of Nitrogen Supplementation on Soil PhysicochemicalProperties and Microbial Diversity in Goji Berry

Goji berry(Lycium barbarum L.)is substantially dependent on nitrogen fertilizer application,which can signifi-cantly enhance fruit yield and Goji berry industrial development in Ningxia,China.This study aimed to analyze the functions of differential nitrogen application rates including low(N1),medium(N2),and high(N3)levels in soil microbial community structure(bacterial and fungal)at 2 diverse soil depths(0-20,20-40 cm)through high-throughput sequencing technology by targeting 16S RNA gene and ITS1&ITS2 regions.All the observed physicochemical parameters exhibited significant improvement(p<0.05)with increased levels of nitrogen and the highest values for most parameters were observed at N2.However,pH decreased(p<0.05)gradually.The alpha and beta diversity analyses for bacterial and fungal communities’metagenome displayed more similarities than differences among all groups.The top bacterial and fungal phyla and genera suggested no obvious(p>0.05)differences among three group treatments(N1,N2,and N3).Furthermore,the functional enrichment analysis demonstrated significant(p<0.05)enrichment of quorum sensing,cysteine and methionine metabolism,and transcriptional machinery for bacterial communities,while various saprotrophic functional roles for fungal communities.Conclusively,moderately reducing the use of N-supplemented fertilizers is conducive to increasing soil nitrogen utilization rate,which can contribute to sustainable agriculture practices through improved soil quality,and microbial community structure and functions.

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.

马铃薯卷叶病毒56kD蛋白基因及3'端非编码区克隆和序列分析

根据已报道的马铃薯卷叶病毒基因组序列，设计合成一对特异性引物。以马铃薯卷叶病毒中国分离株（ＰＬＲＶ－Ｃｈ）的ＲＮＡ为模板，反转录合成了ｃＤＮＡ第一条链，经ＰＣＲ扩增后克隆于ｐＵＣ１９质粒中。进一步用ＰＣＲ鉴定，限制酶切分析和序列分析。结果表明，ＰＬＲＶ－Ｃｈ５６ｋＤ蛋白基因由１５２７个核苷酸组成，编码５０８个氨基酸，３＇端非编码区由１４１个核苷酸组成，与国外报道的苏格兰ＰＬＲＶ－Ｓ、荷兰ＰＬＲＶ－Ｎ、加拿大ＰＬＲＶ－Ｃ、澳大利亚ＰＬＲＶ－Ａ各株系核苷酸序列和氨基酸序列有很高的同源性。５６ｋＤ蛋白基因核苷酸同源率分别为９７．１％、９７．８％、９７．１％和９３．９％，推测的氨基酸同源率分别为９６．２％、９６．４％、９５．８％和９４．６％，非编码区核苷酸同源率分别为９７．９％、９７．２％、９８．６％和９８．６％。

The complete mitogenome of the Chinese bush cricket,Gampsocleis gratiosa(Orthoptera:Tettigonioidea)

The complete mitochondrial genome （mitogenome） of Gampsocleis gratiosa was determined. The 15,929 bp in the size of G. gratiosa mitogenome contains a typical gene content, base composition, and codon usage found in metazoan. All 13 protein coding genes （PCGs） of the G gratiosa mitogenome start with a typical ATN codon. The usual termination codons （TAA and TAG） were found from 10 PCGs. However, the atp6, had4, and had5 had incomplete termination codon （T）. The anticodons of all tRNAs are identical to those observed in Drosophila yakuba and Locusta migratoria, and can be folded in the form of a typical clover leaf structure except for trnS （AGN）. The secondary structure of trnS （AGN） was drawn according with the Steinberg-Cedergren tertiary structure. The A＋T content （67.4%） of the A＋T-rich region is relatively lower among the mitogenome regions, in contrast, it usually contains the highest A＋T content for most insects. Two isolated sequence repeat regions （202 bp） were found in the A＋T-rich region with mapping and secondary structure information.

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.

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.

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.