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.     

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.     

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.     

High‐throughput functional marker assay for detection of Xa/xa and fgr genes in rice (Oryza sativa L.)

We apply CE for high‐throughput analysis of functional markers for marker‐assisted selection in rice. The accuracy, throughput and reproducibility of CE analysis for sequence‐tagged site (STS) and simple sequence repeat (SSR) markers for bacterial blight resistance and aroma genes are demonstrated by using a CE system. Multiplex PCR products displayed well‐differentiated allelic variants using different STS and SSR markers for identification of xa13, Xa21 and fgr genes using the CE system compared to 1.2% agarose gel images. Moreover, consumption of PCR product is much less in the CE system compared to traditional agarose gel systems. Sample consumption is less than 0.1 μL per analysis, thereby conserving samples for further downstream analysis. Out of 29 genotypes in BC₁F₃ generation, 16 plants were found homozygous for all the three genes, viz., xa13, Xa21 and fgr. These homozygous lines can be used as potential donors in rice breeding programmes.     

Identification of novel microsatellite markers <1 Mb from the HBB gene and development of a single‐tube pentadecaplex PCR panel of highly polymorphic markers for preimplantation genetic diagnosis of beta‐thalassemia

Beta (β)‐thalassemia is one of the most common monogenic diseases worldwide. Affected pregnancies can be avoided through preimplantation genetic diagnosis (PGD), which commonly involves customized assays to detect the different combinations of β‐globin (HBB) gene mutations present in couples, in conjunction with linkage analysis of flanking microsatellite markers. Currently, the limited number of reported closely linked markers hampers their utility in indirect linkage‐based PGD for this disorder. To increase the available markers closely flanking the HBB gene, an in silico search was performed to identify all markers within 1 Mb flanking the HBB gene. Fifteen markers with potentially high polymorphism information content (PIC) and heterozygosity values were selected and optimized into a single‐tube pentadecaplex PCR panel. Allele frequencies and polymorphism and heterozygosity indices of each marker were assessed in five populations. A total of 238 alleles were observed from the 15 markers. PIC was >0.7 for all markers, with expected heterozygosity and observed heterozygosity values ranging from 0.74 to 0.90 and 0.72 to 0.88, respectively. Greater than 99% of individuals were heterozygous for at least seven markers, with at least two heterozygous markers on either side of the HBB gene. The pentadecaplex marker assay also performed reliably on single cells either directly or after whole genome amplification, thus validating its use in standalone linkage‐based β‐thalassemia PGD or in conjunction with HBB mutation detection.     

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.     

Development of microsatellite markers in<Emphasis Type="Italic"> Cannabis sativa</Emphasis> for DNA typing and genetic relatedness analyses

Microsatellite markers were developed for Cannabis sativa L. (marijuana) to be used for DNA typing (genotype identification) and to measure the genetic relationships between the different plants. Twelve different oligonucleotide probes were used to screen an enriched microsatellite library of Cannabis sativa in which 49% of the clones contained microsatellite sequences. Characterization of microsatellite loci in Cannabis revealed that GA/CT was the most abundant class of the isolated microsatellites representing 50% overall followed by GTT/CAA, AAG/TTC, and GAT/CTA representing 16%, 15%, and 10%, respectively. Eleven polymorphic STR markers were developed, three derived from dinucleotide motifs and eight from trinucleotide motifs. A total of 52 alleles were detected averaging 4.7 alleles/locus. The expected heterozygosity of the eleven loci ranged between 0.368 and 0.710 and the common probability of identical genotypes was 1.8×10^–7. The loci identified 27 unique profiles of the 41 Cannabis samples. The 11 microsatellite markers developed in this study were found to be useful for DNA typing and for assessing genetic relatedness in Cannabis.     

Population Structure Analysis and Selection of Core Set among Common Bean Genotypes from Jammu and Kashmir,India

Understanding the genetic diversity of a crop is useful for its effective utilization in breeding programmes. For better understanding of the genetic variability in common bean, the first and foremost step is to study its genetic diversity. In the present investigation, 138 genotypes of common bean collected from various regions of Jammu and Kashmir, India, representing major common bean growing areas of this region, were evaluated using 23 SSRs. These SSRs were found highly polymorphic and possess high values for various parameters indicating their high discriminatory power. The average PIC value observed was 0.692, with 0.730 as average gene diversity value, and 0.267 as heterozygosity. Twenty-three SSRs produced a total of 251 alleles. The dendrogram generated with un-weighted neighbour joining cluster analysis grouped genotypes into three main clusters with various degrees of sub-clustering within the clusters. The model-based STRUCTURE analysis using 23 SSR markers identified a population with 3 sub-populations which corresponds to distance-based groupings with average F _ST value and expected heterozygosity of 0.1497 and 0.6696, respectively, within the sub-population, as such high level of genetic diversity was observed within the population. Further, Core Hunter II was used to identify a core set of 96 diverse genotypes. This core set of diverse 96 genotypes is a potential resource for association mapping studies and can be used by breeders as a material to make desirable genetic crosses to generate elite varieties for the fulfilling global market needs. These findings have further implications in common bean breeding as well as conservation programs.     

Characterization of Sulfur-Compounds as Chemotaxonomic Markers in the Essential Oils of Allium Species by Solvent-Free Microwave Extraction and Gas Chromatography–Mass Spectrometry

Allium species were examined to authenticate the chemotaxonomic controversy about these plants by analyzing their extracted compound profiles. Essential oils of various species were isolated using conventional hydro-distillation and solvent-free microwave extraction (SFME). A comparison of the isolation procedures was performed. The presence of sulfur compounds in the Allium genus is a prominent characteristic for their medicinal uses. These components were characterized using two-way hierarchical cluster analysis (HCA) and principal component analysis (PCA). The variation of sulfur-compounds was performed by qualitative analysis of Allium species by gas chromatography–mass spectrometry (GC–MS). 2,4-Dimethyl-5,6-dithia-2,7-nonadienal, 4,6-diethyl-1,2,3,5-tetrathiolane, and 5,7-diethyl-1,2,3,4,6-pentathiepane were revealed as potential chemotaxonomic markers for all of the Alliums examined in this study. These markers may be used to provide improved systematics for other Allium species.     

Simultaneous quantitative analysis of nine constituents in six Chinese medicinal materials from Citrus genus by high‐performance liquid chromatography and high‐resolution mass spectrometry combined with chemometric methods

In this study, we aim to determine the chemical constituents of six Chinese medicinal materials from the Citrus genus using high‐performance liquid chromatography and high‐resolution mass spectrometry. Eight flavonoids and one coumarin were identified and further quantified as marker substances by high‐performance liquid chromatography method. The separation was performed on an Agilent TC‐C₁₈ column with 0.1% formic acid and acetonitrile as the mobile phase under gradient elution. The analytical method was fully validated in terms of linearity, sensitivity, intra‐ and inter‐day precision and repeatability, limit of detection, limit of quantitation, and recovery. It was subsequently applied to evaluate the quality of 103 batches of the Chinese medicinal materials from the Citrus genus. In addition, the principal constituent analysis was used to compare the samples of different species from the Citrus genus leading to successful classification of the samples in accordance with their origins. It was found that the contents of nine constituents varied greatly in different ripening stages and varieties of the samples from the Citrus genus. In addition, neoeriocitrin and 5,7‐dimethoxycoumarin were determined as two unique constituents of ‘Zhiqiao’ and ‘Foshou’, respectively. In conclusion, this study provides a chemical basis for quality control of Chinese medicinal materials from the Citrus genus.     

Mining of EST-SSR Markers of Musa and Their Transferability Studies Among the Members of Order the Zingiberales

Expressed sequence tags (ESTs) databases of 11 Musa complementary DNA libraries were retrieved from National Center of Biotechnology Information and used for mining simple sequence repeats (SSRs). Out of 21,056 unique ESTs, SSR regions were found only in 5,158 ESTs. Among these SSR containing ESTs, the occurrence of trinucleotide repeats are the most abundant followed by mono-, di-, tetra-, hexa-, and pentanucleotides. Moreover, this study showed that the rate of class II SSRs (<20 nucleotides) was higher than the class I SSRs (<20 nucleotides), and proportion of class I and II SSRs as abundant for tri-repeats. As a representative sample, primers were synthesized for 24 ESTs, carrying >12 nucleotides of SSR region, and tested among the various genomic group of Musa accessions. The result showed that 88 % of primers were functional primers, and 43 % are showing polymorphism among the Musa accessions. Transferability studies of Musa EST-SSRs among the genera of the order Zingiberales exhibited 100 and 58 % transferability in Musaceae and Zingiberaceae, respectively. The sequence comparison of SSR regions among the different Musa accessions confirmed that polymorphism is mainly due to the variation in repeat length. High percentage of cross-species, cross-genera, and cross-family transferability also suggested that these Musa EST-SSR markers will be a valuable resource for the comparative mapping by developing COS markers, in evolutionary studies and in improvement of the members of Zingiberaceae and Musaceae.     

Quantitative determination of isoquinoline alkaloids and chlorogenic acid in Berberis species using ultra high performance liquid chromatography with hybrid triple quadrupole linear ion trap mass spectrometry

Berberis species are well known and used extensively as medicinal plants in traditional medicine. They have many medicinal values attributable to the presence of alkaloids having different pharmacological activities. In this study, a method was developed and validated as per international conference on harmonization guidelines using ultra high performance liquid chromatography with hybrid triple quadrupole‐linear ion trap mass spectrometry operated in the multiple reaction monitoring mode for nine bioactive compounds, including protoberberine alkaloids, aporphine alkaloids and chlorogenic acid. This method was applied in different plant parts of eight Berberis species to determine variations in content of nine bioactive compounds. The separation was achieved on an ACQUITY UPLC CSH? C₁₈ column using a gradient mobile phase at flow rate 0.3 mL/min. Calibration curves for all the nine analytes provided optimum linear detector response (with R² ≥0.9989) over the concentration range of 0.5–1000 ng/mL. The precision and accuracy were within RSDs ≤2.4 and ≤2.3%, respectively. The results indicated significant variation in the total contents of the nine compounds in Berberis species.     

虾夷马粪海胆微卫星标记制备及对3个养殖群体的遗传多样性分析

通过磁珠富集法分离获得了虾夷马粪海胆微卫星DNA序列160个.其中完美型108个(67.50%),非完美型33个(20.63%),复合型19个(11.88%),微卫星DNA序列最多重复次数达到107次.根据这些微卫星DNA序列的两翼序列,采用Primer 5软件设计出虾夷马粪海胆的微卫星引物,通过筛选,采用其中的12对微卫星DNA标记对大连凌水群体(DL)、大连獐子岛群体(DZ)、山东荣城群体(SR)3个虾夷马粪海胆养殖群体的遗传多样性进行分析,共获得73个等位基因,不同引物获得的等位基因数为1~11个,片段大小为84~362 bp.除SUX001位点外,其余位点的PIC值在0.264 0~0.670 5之间,3个群体的平均观测杂合度分别为0.4618(DL),0.437 5(SR)和0.434 0(DZ),平均期望杂合度分别为0.502 6(DL),0.507 9(SR)和0.449 4(DZ).Hardy-Weinberg平衡分析显示,73%的被检测位点显著偏离平衡,F-检验显示4个位点的Fst值低于0.05,表明群体间存在一定程度的分化.群体间遗传相似性系数、遗传距离及UPGMA聚类分析表明大连獐子岛群体(DZ)与大连凌水群体(DL)群体亲缘关系较近,二者与山东荣成群体(SR)亲缘关系较远.对深入了解我国虾夷马粪海胆养殖群体遗传结构特征及其种质资源状况具有重要意义.     

HPLC Fingerprint Characteristics of Active Materials of Garlic and Other Allium Species

The bioactive molecules of garlic are classified according to their enzymatic activities as either alliinase activators or alliinase inactivators. The alliinase activation reaction system is mainly composed of sulfur-containing compounds, whereas the alliinase inactivation system is mainly composed of amino acid-based materials. The purpose of this study was to develop an effective way to digitally express features of complex active compounds of garlic as a basis for quality control. HPLC was used to develop the fingerprints of plants from different Allium species and different geographical regions according to whether the compounds were sulfur containing or based on amino acids. Using the Shannon equation, I values of sulfur-containing compounds from garlic ranged from 3.55 to 3.94, whereas I values of sulfur-containing compounds from other Allium plants, onions, leek, and Welsh onion, ranged from 3.38 to 3.53. The I values of amino acid-based compounds from garlic ranged from 3.67 to 3.91, whereas I values of these compounds from other Allium plants ranged from 3.88 to 3.99. This method effectively distinguished garlic from different species of Allium plants. This method also provided a way to digitally monitor the presence of complex active compounds of garlic and may allow evaluation of quality. This method may also provide a theoretical basis for quality control of bioactive compounds from other medicinal plants.     

An iboga alkaloid chemotaxonomic marker from endemic Tabernaemontana ternifolia with antitubercular activity

Abstract

Coronaridine (1) was isolated from the CH₂Cl₂ root extract of Tabernaemontana ternifolia. The structure of 1 was established from 1D- and 2D-NMR and HR-ESIMS experiments, and by comparison with reported spectroscopic data. To date, this is the first report of compound 1 from T. ternifolia, introduced as new Tabernaemontana species from Philippines in 2005 on the basis of morphological characters. Coronaridine, an iboga-type indole alkaloid, has been isolated from over 50 Tabernaemontana species and can thus be inferred as a chemotaxonomic marker of the genus. T. ternifolia has a distinct arrangement of leaves not known in the genus, but is variable in other genera. Its isolation from endemic T. ternifolia establishes its position in the genus and supports the claim that coronaridine is a chemical marker of the genus Tabernaemontana. Interestingly, coronaridine exhibited relatively weak activity against Mycobacterium tuberculosis H₃₇Rv (MIC 82.64?μg/mL) (Rifampicin MIC 0.05?μg/mL).     

Screening of Bletilla striata,Bletilla ochracea and Oreorchis foliosa differential metabolites based on metabolomics

As a representative medicinal plant in the Orchidaceae, Bletilla striata plays a variety of pharmacological roles in the clinic. However, the emergence of counterfeit species is affecting the basic medicinal materials source identification process, for which Bletilla ochracea and Oreorchis foliosa of the Orchidaceae are two representative species. For this study, 13 representative B. striata samples, three B. ochracea samples and three O. foliosa samples were selected for the systematic determination of polysaccharide yields and monosaccharide composition, and further detection of secondary metabolites by HPLC–MS. The results revealed that there was a significant difference in the yields of polysaccharides between B. striata and B. ochracea (p = 0.006). Although the polysaccharides of both species were composed of glucose and mannose, the molar ratio of the two monosaccharides was different, suggesting that the structures of the polysaccharides were different. The metabolomics results showed that there were no differences in the types of metabolites between B. striata and B. ochracea; however, there were differences in the contents of these metabolites. Although there was no significant difference in the polysaccharide yields of B. striata and O. foliosa (p = 0.074) and the monosaccharide composition was the same (glucose and mannose), many different metabolites were screened out between them: six compounds such as C₃₆H₃₄O₁₁ existed only in B. striata, while substance C₃₉H₅₄O₂₂ was unique to O. foliosa. Therefore, based on the analysis of the polysaccharide content and monosaccharide composition, combined with phase metabolomics research, a preliminary distinction between B. striata, B. ochracea and O. foliosa was achieved.     

Interaction of Phosphate with Iron(III) in Acidic Medium,Equilibrium and Kinetic Studies

Equilibrium reactions of iron(III) with phosphate were studied spectrophotometrically by UV-Vis in the pH range of ～ 1.0-2.20. The STAR-94 Program was used to determine the number of absorbing species as well as the stoichiometries and formation constants of the complex species. Some literature values were further confirmed and new values of different stoichiometries were obtained. The kinetics and mechanism of Fe(III) with phosphate were studied in acidic medium. The reactive phosphate species were found to be only H₃PO₄ and H₂PO^? ₄ and for Fe(III) were only Fe³⁺, FeOH²⁺ and Fe(OH)⁺ ₂. The observed rate constants were pH as well as T_phos (total concentration of phosphate) dependent, i.e. K_obs,i = A _i + B _i T_phos + C _i T² _phos (at a given pH).     

Iron(II)-bis (salicyladimine) Schiff-base complexes catalyzed decomposition of hydrogen peroxide

The tetradentate Schiff-base ligands, N,N′-bis(salicylidene)-ethylenediamine (Salen), N,N′-bis(salicylidene) butylenediamine (Salbut), and N,N′-bis(salicylidene)-o–phenylenediamine, (sal-o-phen) are very strongly sorbed by cation exchange resin (Dowex-50W) with Fe²⁺ ions as a counter ion, forming stable complexes. The kinetics of the catalytic decomposition of H₂O₂ using these complexes was studied in ethanolic medium. The reaction was first-order with salen and sal-o-phen and second-order with salbut with respect to [H₂O₂]. The rate of the H₂O₂ decomposition increased either from salen to salbut or from salen to sal-o-phen. Also, the k (per g dry resin) values decreased with increasing both the particle size and the degree of resin cross-linkage. The active species formed at the beginning of the reaction, had an inhibiting effect on the reaction rate. The corresponding activation parameters were calculated from a least-squares fit of the temperature dependence of the rate constant. A reaction mechanism is proposed. © 1994 John Wiley & Sons, Inc.     

A new differential scanning calorimetry based approach for the characterization of peracid resin

Differential scanning calorimetry was used to trace the heat of decomposition of the peracid groups (ΔH_d) in the oxidized EVA-g-AA resin. From the correlation between ΔH_d and the oxidation capacity measured by iodometry, it was found that 35 ± 5 cal of energy evolved per miliequivalent of peracid group decomposed. The ΔH_d values are also useful in finding the optimum condition for oxidation of acid groups and can be used to investigate the distribution of active peracid groups across the matrix. The stability and the activation energy of decomposition of grafted type peracid resin were also studied, where the activation energy of decomposition was calculated to be 13.4 kcal/mol.     

The genus Machaerium (Fabaceae): taxonomy,phytochemistry, traditional uses and biological activities

Machaerium, in the family Fabaceae, predominantly is a genus of a Neotropical distribution of trees, shrubs, and lianas occurring from southern Mexico to Brazil and northern Argentina and as far as South America. Several Machaerium species are widely used in traditional medicine and are considered to have multiple medicinal properties. This review aims to provide up-to-date and comprehensive information on the taxonomy, phytochemistry, traditional uses and biological activities of plants in the genus Machaerium.