Development and Characterization of Microsatellite Markers (SSR) in Sesamum (Sesamum indicum L.) Species

Microsatellites, also known as simple sequence repeats (SSRs), are the class of repetitive DNA sequences present throughout the genome of many plant and animal species. Recent advances in molecular genetics had been the introduction of microsatellite markers to investigate the genetic structuring of natural plant populations. We have employed an enrichment strategy for microsatellite isolation by using multi-enzymes digestion, microsatellite oligoprobes, and streptavidin magnetic beads in Sesamum (Sesamum indicum L.). More than 200 SSR motifs were detected (SSR motifs ??2 repeat units or 6?bp); 80?% of the clones contained SSR motifs. When regarding SSRs with four or more repeat units and a minimum length of 10?bp, 132 of them showed repeats. Eighteen SSR markers were initially characterized for optimum annealing temperature using a gradient PCR technique. Among the 18 SSR markers characterized, five were found to be polymorphic and used to analyze 60 Sesamum germplasm accessions. The maximum number of alleles detected was four with a single primer and the least number of two alleles with three primers with an average PIC value of 0.77. SSRs are a valuable tool for estimating genetic diversity and analyzing the evolutionary and historical development of cultivars at the genomic level in sesame breeding programs.     

SSR-Based DNA Fingerprinting and Diversity Assessment Among Indian Germplasm of <Emphasis Type="Italic">Euryale ferox</Emphasis>: an Aquatic Underutilized and Neglected Food Crop

Euryale ferox is native to Southeast Asia and China, and it is one of the important aquatic food crops propagated mostly in eastern part of India. The aim of the present study was to characterize and evaluate the genetic diversity of ex situ collections of E. ferox germplasm from different geographical states of India using microsatellite (simple sequence repeats (SSRs)) markers. Ten SSR markers were analyzed to assess DNA fingerprinting and genetic diversity of 16 cultivated germplasm of E. ferox. Total 37 polymorphic alleles were recorded with an average of 3.7 allele frequency per primer. The polymorphic information content value varied from 0.204 to 0.735 with mean of 0.448. A high range of heterozygosity (Ho 0.228; He 0.512) was detected in the present study. The neighbor-joining (N-J) tree and the principle coordinate analysis showed that the germplasm divided in to three main clusters. The results of the present investigation comply that SSR markers are effective for computing genetic assessment of genetic diversity and similarity with classifying cultivated varieties of E. ferox. Evaluation of genetic diversity among Indian E. ferox germplasm could provide useful information for genetic improvement.     

Construction of new EST-SSRs for Fusarium resistant wheat breeding

Surveying Fusarium resistance in wheat with easy applicable molecular markers such as simple sequence repeats (SSRs) is a prerequest for molecular breeding. Expressed sequence tags (ESTs) are one of the main sources for development of new SSR candidates. Therefore, 18.292 publicly available wheat ESTs were mined and genotyping of newly developed 55 EST-SSR derived primer pairs produced clear fragments in ten wheat cultivars carrying different levels of Fusarium resistance. Among the proved markers, 23 polymorphic EST-SSRs were obtained and related alleles were mostly found on B and D genome. Based on the fragment profiling and similarity analysis, a 327 bp amplicon, which was a product of contig 1207 (chromosome 5BL), was detected only in Fusarium head blight (FHB) resistant cultivars (CM82036 and Sumai) and the amino acid sequences showed a similarity to pathogen related proteins. Another FHB resistance related EST-SSR, Contig 556 (chromosome 1BL) produced a 151 bp fragment in Sumai and was associated to wax2-like protein. A polymorphic 204 bp fragment, derived from Contig 578 (chromosome 1DL), was generated from root rot (FRR) resistant cultivars (2–49; Altay2000 and Sunco). A total of 98 alleles were displayed with an average of 1.8 alleles per locus and the polymorphic information content (PIC) ranged from 0.11 to 0.78. Dendrogram tree with two main and five sub-groups were displayed the highest genetic relationship between FRR resistant cultivars (2–49 and Altay2000), FRR sensitive cultivars (Seri82 and Scout66) and FHB resistant cultivars (CM82036 and Sumai). Thus, exploitation of these candidate EST-SSRs may help to genotype other wheat sources for Fusarium resistance.     

RAPD and ISSR Marker-Based Comparative Evaluation of Genetic Diversity Among Indian Germplasms of <Emphasis Type="Italic">Euryale ferox</Emphasis>: an Aquatic Food Plant

Euryale ferox Salisbury is an important aquatic food plant cultivated largely in eastern India. E. ferox is a monotypic genus, and breeding programmes have mostly relied on the variability present in the primary gene pool. Knowledge of the genetic structure of the population is limited, and there are very few reports available on the genetic diversity of E. ferox. In this study, comprehensive research on the genetic diversity of 16 germplasms of E. ferox was carried out using random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers. Out of 320 RAPD and 95 ISSR primers screened initially, 61 primers (40 RAPD and 21 ISSR) gave reproducible bands and were selected for further work. Amplification of the 40 RAPD primers gave 533 polymorphic bands with an average of 13.32 polymorphic bands per primer. The percentage of polymorphism ranged from 37.5 to 100, with an average of 88.3 %. The 21 ISSR primers produced 259 bands, of which 214 were polymorphic, with an average of 10.19 polymorphic bands per primer. The percentage of polymorphism using ISSR primers ranged from 50 to 100, with a mean of 82.6 %. Jaccard’s coefficient ranged from 0.45 to 0.69 (RAPD), 0.50 to 0.77 (ISSR) and 0.48 to 0.71 (RAPD and ISSR). Molecular characterization of different germplasms of E. ferox not only is essential for its conservation but also can be used in further breeding programmes.     

Development and Characterization of EST-SSR Markers from <em>Scapharca broughtonii</em> and Their Transferability in <em>Scapharca subcrenata</em> and <em>Tegillarca granosa</em>

Twenty-five novel EST-derived simple sequence repeat (EST-SSR) markers were developed in the ark shell Scapharca broughtonii. Polymorphisms of these EST-SSR markers were evaluated in 48 wild individuals collected from Shidao, Shandong Province, China. A total of 202 alleles were detected at 25 loci. The numbers of alleles per locus ranged from 4 to 14, with an average of 8.08. The observed and expected heterozygosities varied from 0.2917 to 1.000 and from 0.3570 to 0.9002, respectively. After sequential Bonferroni correction for multiple tests, only one locus was found to deviate from Hardy-Weinberg equilibrium. Twenty-five EST-SSR markers showed a high rate of across-species transferability (100%) in Scapharca subcrenata and a low rate of across-genus transferability (20%) in Tegillarca granosa. These EST-SSRs will be helpful for QTL mapping, molecular breeding and investigation of population genetic diversity in ark shell S. broughtonii and other Scapharca species.     

Metabolite Profiling of Gardenia jasminoides Ellis In Vitro Cultures with Different Levels of Differentiation

Gardenia jasminoides Ellis is an aromatic and medicinal plant of high economic value. Much research has focused on the phytochemistry and biological activities of Gardenia fruit extracts; however, the potential of the Gardenia plant in vitro cultures used as mass production systems of valuable secondary metabolites has been understudied. This paper presents data on metabolite profiling (GC/MS and HPLC), antioxidant activities (DPPH, TEAC, FRAP, and CUPRAC), and SSR profiles of G. jasminoides plant leaves and in vitro cultures with different levels of differentiation (shoots, callus, and cell suspension). The data show strong correlations (r = 0.9777 to r = 0.9908) between antioxidant activity and the concentrations of chlorogenic acid, salicylic acid, rutin, and hesperidin. Eleven co-dominant microsatellite simple sequence repeats (SSRs) markers were used to evaluate genetic variations (average PIC = 0.738 ± 0.153). All of the investigated Gardenia in vitro cultures showed high genetic variabilities (average Na = 5.636 ± 2.157, average Ne = 3.0 ± 1.095). This is the first report on a study on metabolite profiles, antioxidant activities, and genetic variations of G. jasminoides in vitro cultures with different levels of differentiation.     

The frequency of poly(G) tracts in the human genome and their use as a sensor of DNA damage

Tandem repeats of short DNA sequences are commonly found in human DNA. These simple sequence repeats or microsatellites are highly polymorphic in the human genome. Since the anti-tumour agent cisplatin preferentially forms DNA adducts at runs of consecutive guanine nucleotides (poly(G)), the position and frequency of occurrence of poly(G) sequences in the updated human genome was investigated. There are more runs of consecutive guanines than would be expected by random chance. This especially true for poly(G) sequences longer than approximately n = 9. A plot of poly(G) length against log(observed/expected) frequency produced a straight line for n > 9. A similar observation was also found for poly(A) DNA sequence repeats. This data implied that the increase in observed/expected frequency is directly related to length of DNA repeat. It was proposed that long runs of consecutive guanine nucleotides could be a sensitive sensor of cellular DNA damage since a number of DNA damaging agents cause lesions at poly(G) sequences.     

Diversity of Volatile Compounds in Ten Varieties of Zingiberaceae

Zingiberaceae plants are distributed in the tropical and subtropical regions of the world, being used in many famous medicinal materials. Meanwhile, some Zingiberaceae plants are important horticultural flowers because they are green all year round and have special aromas. To conduct an extensive investigation of the resources of Zingiberaceae plants, the volatile compounds of ten species of Zingiberaceae were extracted and analyzed by GC–MS, including Costus comosus var. bakeri (K.Schum.) Maas, Curcuma rubescens Roxb., Curcuma aeruginosa Roxb., Curcuma attenuata Wall., Hongfengshou, Hedychium coronarium Koeng, Zingiber zerumbet (L.) Smith, Hedychium brevicaule D. Fang, Alpinia oxyphylla Miq., and Alpinia pumila Hook.F. A total of 162 compounds were identified, and most of those identified were monoterpenes and sesquiterpenes. (E)-labda-8(17),12-diene-15,16-dial, n-hexadecanoic acid, 4-methoxy-6-phenethyl-2H-pyran-2-one, and L-β-pinene were found in high concentrations among the plants. These ten species of Zingiberaceae contained some of the same volatiles, but their contents were different. Pharmacological effects may be associated with the diversity of volatiles in these ten plants.     

Molecular Characterization of Mosquitocidal Toxin (Surface Layer Protein,SLP) from <Emphasis Type="Italic">Bacillus cereus</Emphasis> VCRC B540

A marine Bacillus cereus (VCRC B540) with mosquitocidal effect was recently reported from red snapper fish (Lutjanus sanguineous) gut and surface layer protein (S-layer protein, SLP) was reported to be mosquito larvicidal factor. In this present study, the gene encoding the surface layer protein was amplified from the genomic DNA and functionally characterized. Amplification of SLP-encoding gene revealed 1,518 bp PCR product, and analysis of the sequence revealed the presence of 1482 bp open reading frame with coding capacity for a polypeptide of 493 amino acids. Phylogenetic analysis revealed with homology among closely related Bacillus cereus groups of organisms as well as Bacillus strains. Removal of nucleotides encoding signaling peptide revealed the functional cloning fragment of length 1398 bp. Theoretical molecular weight (51.7 kDa) and isoelectric point (5.99) of the deduced functional SLP protein were predicted using ProtParam. The amplified PCR product was cloned into a plasmid vector (pGEM-T), and the open reading frame free off signaling peptide was subsequently cloned inpET-28a(+) and expressed in Escherichia coli BL21 (DE3). The isopropyl-β-D-thiogalactopyranoside (IPTG)-induced recombinant SLP was confirmed using western blotting, and functional SLP revealed mosquito larvicidal property. Therefore, the major findings revealed that SLP is a factor responsible for mosquitocidal activity, and the molecular characterization of this toxin was extensively studied.     

Identification of Heat-Related ESTs in Moth Bean Through Suppression Subtraction Hybridization

Moth bean (Vigna aconitifolia (Jacq.) Marechal), an important grain-legume crop grown in hot desert regions of Thar, under scorching sun rays, was investigated for heat tolerance at molecular level. In the present study, we constructed a forward suppression subtractive hybridization (SSH) cDNA library of heat tolerant genotype RMO-40 to identify genes expressing under delayed response to elevated temperature. Heat induction was carried out by exposing 14-day-old seedlings to elevated temperature of 42 °C for 30 min. A total of 125 unigenes (33 contigs and 92 singletons) were derived by cluster assembly and sequence alignment of 200 ESTs; out of 125 unigenes, 21 (16 %) were found to be novel to moth bean. Gene ontology functional classification terms were retrieved for 98 (78.4 %) unigenes of which 73 (58.4 %) ESTs were functionally annotated (GO consensus) where 19 unigenes were annotated with 11 enzyme commission (EC) codes and were mapped to 25 different KEGG pathways. We have identified a majority of heat-shock proteins (constituting 35 % of the present library) aiding heat stress tolerance to moth bean. An expression level of 22 ESTs generated from the above SSH cDNA library was studied through semiquantitative RT-PCR assay simultaneously under 5 and 30 min of heat stress at 42 °C.     

Evaluation of ultrahigh-performance liquid chromatography columns for the analysis of unmodified and antisense oligonucleotides

Ultrahigh-performance liquid chromatography has been used for the separation and analysis of unmodified and modified antisense oligonucleotides. For this reason, we tested various columns of low particle sizes in our analysis of unmodified and phosphorothioate oligonucleotides. The influence of both the type and concentration of ion-pair reagent on the retention of the studied biomolecules was tested. The developed methods were used for separation of unmodified oligonucleotides and to determine antisense oligonucleotides in human serum samples. The results proved that octadecyl and phenyl columns are the most selective in the resolution of oligonucleotides which differ in the position of single nucleotides in the sequence. The phenyl column was selected and applied for the analysis of phosphorothioate oligonucleotides in serum samples. The calibration plots showed good linearity within the test concentration ranges. The intra-day CV of the calibration curve slopes was in the range of 1.6 to 4.2 %. The limits of detection (LODs) were in the range of 0.11–0.16 μg mL^?1, while the limit of quantification (LOQ) values were between 0.35 and 0.51 μg mL^?1. Figure

Determination of antisense phosphorothioate oligonucleotides in serum     

Inter-Simple Sequence Repeat (ISSR) Marker Analysis in Parkia timoriana (DC.) Merr. Populations from Northeast India

Genetic variation in three populations of Parkia timoriana (DC.) Merr. grown in the Manipur state of northeast India was analysed using inter-simple sequence repeat (ISSR) markers. A total of 30 individual trees representing three populations were sampled and studied using 22 University of British Columbia (UBC set no. 9) primers in the present study. Of the total 22 primers, 19 primers produced distinct, reproducible and well-resolved fragments. Overall, a total number of 111 fragments were generated by the 19 primers and of which, 51 were polymorphic (45.94 %). The average number of loci and polymorphic loci generated per primer were 5.84 and 2.68, respectively. The genetic variation generated by ISSR markers within the three populations studied ranges from 33.33 to 18.92 %. The overall genetic differentiation (Gst) among populations was estimated to be 0.29, and the number of gene flow (Nm) was estimated to be 1.23 per generation between populations. Of the total genetic variance, 70.04 % was attributed to within-population diversity while 4.72 % differences to the among-populations. The genetic similarity across the individuals belonging to the three populations was represented by the dendrogram showing the grouping of the individuals into three major groups which is also supported by the principle component analysis. The present finding asserts the effectiveness of ISSR procedure for assessing genetic variations of P. timoriana populations and provides valuable genetic information that can be utilized for breeding and conservation strategies.     

Synthesis of musafluorone: a naphthoxanthenone isolated from Musa acuminata

5-Methoxy-3H-naphtho[2,1,8-mna]xanthen-3-one (musafluorone, 1), the only naphthoxanthenone reported so far from Musaceae, was synthesized starting from 2-naphthol in nine steps and resulted in an overall yield of 3%. Grignard addition of phenylmagnesium bromide to 4-methoxyperinaphthenone afforded the corresponding 4-methoxy-9-phenylphenalenone which, after epoxidation and methyl transposition, was subjected to a photochemical cyclization procedure to furnish 1.     

微流控芯片电泳-多重聚合酶链反应法同时测定多个单碱基多态性位点

以微流控芯片电泳为检测平台,建立了多重PCR扩增法同时测定多个单碱基多态性(SNP)位点的方法。先通过PCR扩增得一段含所有待测SNP位点的长片段;用限制性内切酶消化成短片段,再将酶切反应产物与脱氧核糖核酸适配器(DNAadapter)相连;以连接产物为模板,分成两管,分别用n条等位基因特异性引物和一条通用引物进行n重PCR扩增;最后用微流控芯片电泳法分离PCR扩增产物,根据两管扩增产物的芯片电泳图谱中扩增片段的大小判断SNP的类型。以细胞色素P4502D6(CYP2D6)基因中的5个SNP位点(100C>T、1661G>C、1758G>T、2470T>C和2850C>T)为检测对象,考察了各等位基因特异性引物之间的相互影响和扩增反应的特异性,采用微流控芯片电泳法成功测定了20名健康中国人的CYP2D6基因中5个SNP位点的基因多态性,与聚合酶链反应-限制性片段长度多态性法(PCR-RFLP)测定结果完全一致。     

The detection of T-Nos,a genetic element present in GMOs,by cross-priming isothermal amplification with real-time fluorescence

An isothermal cross-priming amplification (CPA) assay for Agrobacterium tumefaciens nopaline synthase terminator (T-Nos) was established and investigated in this work. A set of six specific primers, recognizing eight distinct regions on the T-Nos sequence, was designed. The CPA assay was performed at a constant temperature, 63 °C, and detected by real-time fluorescence. The results indicated that real-time fluorescent CPA had high specificity, and the limit of detection was 1.06?×?10³ copies of rice genomic DNA, which could be detected in 40 min. Comparison of real-time fluorescent CPA and conventional polymerase chain reaction (PCR) was also performed. Results revealed that real-time fluorescent CPA had a comparable sensitivity to conventional real-time PCR and had taken a shorter time. In addition, different contents of genetically modified (GM)-contaminated rice seed powder samples were detected for practical application. The result showed real-time fluorescent CPA could detect 0.5 % GM-contaminated samples at least, and the whole reaction could be finished in 35 min. Real-time fluorescent CPA is sensitive enough to monitor labeling systems and provides an attractive method for the detection of GMO.

Quantitative analysis of multiple genes’ expressions based on a novel competitive RT-PCR assay

We established a novel gene expression analysis platform, Multiplex Competitive RT-PCR Using Fluorescent Universal Primers (MCF-PCR), to study multi-gene expression patterns simultaneously. This platform combines fluorescent universal primers, multiplex competitive RT-PCR, and capillary electrophoretic separation, which ensures MCF-PCR a reliable, medium-throughput, cost-effective technology for gene expression profiling. With cloned standard DNAs, the detection limits, precision, and sensitivity of MCF-PCR were evaluated and compared with that of the assay without adding competitive templates and real-time PCR, respectively. The results showed that detection limit was 3.125?×?10³ to 3.2?×?10⁶ copies, and 10 % copy differences between two samples can be detected by MCF-PCR. To validate MCF-PCR, we analyzed expression profile of five genes in interleukin (IL)-4/IL-13 pathway in peripheral blood of 20 healthy adults and 20 allergic dermatitis patients; three genes including IL-4, IL-13, and STAT6 were found differentially expressed in the two sample groups, which maybe key players in IL-4/IL-13 immunological signaling pathway and need further function analysis.

Detecting authorized and unauthorized genetically modified organisms containing vip3A by real-time PCR and next-generation sequencing

The growing number of biotech crops with novel genetic elements increasingly complicates the detection of genetically modified organisms (GMOs) in food and feed samples using conventional screening methods. Unauthorized GMOs (UGMOs) in food and feed are currently identified through combining GMO element screening with sequencing the DNA flanking these elements. In this study, a specific and sensitive qPCR assay was developed for vip3A element detection based on the vip3Aa20 coding sequences of the recently marketed MIR162 maize and COT102 cotton. Furthermore, SiteFinding-PCR in combination with Sanger, Illumina or Pacific BioSciences (PacBio) sequencing was performed targeting the flanking DNA of the vip3Aa20 element in MIR162. De novo assembly and Basic Local Alignment Search Tool searches were used to mimic UGMO identification. PacBio data resulted in relatively long contigs in the upstream (1,326 nucleotides (nt); 95 % identity) and downstream (1,135 nt; 92 % identity) regions, whereas Illumina data resulted in two smaller contigs of 858 and 1,038 nt with higher sequence identity (>99 % identity). Both approaches outperformed Sanger sequencing, underlining the potential for next-generation sequencing in UGMO identification.

Cloning,Expression, and Characterization of a Milk-Clotting Aspartic Protease Gene (Po-Asp) from Pleurotus ostreatus

An aspartic protease gene from Pleurotus ostreatus (Po-Asp) had been cloned based on the 3′ portion of cDNA in our previous work. The Po-Asp cDNA contained 1,324 nucleotides with an open reading frame (ORF) of 1,212 bp encoding 403 amino acid residues. The putative amino acid sequence included a signal peptide, an activation peptide, two most possible N-glycosylation sites and two conserved catalytic active site. The mature polypeptide with 327 amino acid residues had a calculated molecular mass of 35.3 kDa and a theoretical isoelectric point of 4.57. Basic Local Alignment Search Tool analysis showed 68–80 % amino acid sequence identical to other basidiomycetous aspartic proteases. Sequence comparison and evolutionary analysis revealed that Po-Asp is a member of fungal aspartic protease family. The DNA sequence of Po-Asp is 1,525 bp in length without untranslated region, consisting of seven exons and six introns. The Po-Asp cDNA without signal sequence was expressed in Pichia pastoris and sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated the molecular mass of recombinant Po-Asp was about 43 kDa. The crude recombinant aspartic protease had milk-clotting activity.