Molecular Marker Techniques Research Articles

Evaluation of genetic diversity and population structure of polygonati rhizoma germplasms: implications for better crop development and conservation of a traditional Chinese medicine

Polygonati rhizoma (PR) is an important and widely used product in Chinese traditional medicine and edible goods. The time-consuming nature of breaking dormancy in both rhizomes and seeds means that improving variety selection is limited to collection, identification, and selection of germplasms. In this study, we used two DNA-based molecular marker techniques—inter simple sequence repeat (ISSR) and start codon targeted (SCoT)—to assess the genetic diversity and population structure among PR source plants collected from 47 different regions and belonging to 12 populations (P1–P12). For molecular markers analysis, 15 ISSR and 10 SCoT markers were tested. Total number of 159 fragments (150–4000 bp) were amplified based on ISSR analysis with a range from 6 to 17 bands, 153 of them were polymorphic, ranging from 97.27 to 100%. For SCoT analysis, 164 polymorphic bands (150–5000 bp) were observed, varying from 14 to 19 bands for each primer. Nei’s genetic diversity analysis showed that the highest value was found in P11 for ISSR and P4 for SCoT markers. The highest Nei’s genetic diversity value was observed in P5 for combined markers and the low in P2. Nei’s dendrogram constructed with combined markers indicated a 75–89% of genetic similarity coefficient among populations. Population structure analysis revealed an optimum number of three groups, the same as their geographical distribution. This knowledge on PR genetic diversity can be used in future breeding programs, genetic improvement, product enhancement, and germplasms conservation.

A Brief Review of Molecular Markers to Analyse Medically Important Plants

Suitable identification and characterisation of plants using in medicine are necessary for conservation plant resources, investigations of genes associated with desirable traits, and understanding of evolutionary relationships. Therefore, various molecular marker techniques such as RAPD, AFLP, SSR and ISSR, SNP, SCoT, ITS and SCAR have been improved to provide detail information about genomes, which were not previously possible with only phenotypic methods. This brief review represents usage of these markers for molecular diversity analyses of medically important plants.

Genetic diversity of 6 species in Polygonatum by SSR marker

Polygonatum is a genus of the perennial herb family Liliaceae, with great potential in food, medicine and other field. In this study, genetic diversity and cluster structure analysis of 6 species in Polygonatum were investigated the molecular marker technique of simple sequence repeat (SSR). A total of 49 SSR makers were used to study genetic diversity and population structure within 60 germplasm resources which obtained from 38 counties and cities in 14 provinces of China. A total of 211 alleles were identified and the number of alleles ranged from 2 to 10, with an average of 4.306 1 alleles per SSR. The range of polymorphism information content (PIC) varied from 0.731 8 to 0.031 7, with the average of 0.309 6. The cluster analysis classified 60 germplasm resources into four defined groups at the genetic distance value of 0.26, among which most species with relatives were clustered into the same group. Extraordinarily, there were 6 germplasm resources clustered into other species, indicating that the classification of inter-genus and geographical distribution was crossed in Polygonatum. The genetic diversity index of the 4 geographical populations from high to low was: Western region, Central China, Southern China, Eastern China. The population structure analysis, also indicating divided the entire collection into four groups, which was similar to the assignment pattern of cluster structure analysis. These results suggested that the Polygonatum germplasm resources used in this study is rich in relatively high genetic diversity with large variation range, relatively fuzzy boundaries of species. It appeared the phenomenon that there is a difference decreases between the alternate leaf system and the rotation leaf system. The genetic diversity in the western region was higher than that in other regions, and the western region may be the origin center of the genus Polygonatum.

Study of genetic variation and morphological traits in 25 melon cultivars by using molecular marker techniques

Study of genetic variation and morphological traits in 25 melon cultivars by using molecular marker techniques

Detection of HERV-K6 and HERV-K11 transpositions in the human genome.

Mobile genetic elements classed as transposons comprise an estimated 45% of the human genome, and 8% of these elements are human endogenous retroviruses (HERVs). Endogenous retroviruses are retrotransposons, containing 5' and 3' long terminal repeat sequences and encoding envelope, group-specific antigen and DNA polymerase proteins. The aim of the present study was to analyse genome integration polymorphisms of HERV type K member 6 (HERV-K6) and HERV-K11 by using the retrotransposon based molecular marker technique, inter-retrotransposon amplified polymorphism (IRAP). For this purpose, blood samples of 18 healthy individuals within the age range of 10-79 years (10 females and 8 males) were collected, genomic DNAs were isolated and IRAP-polymerase chain reaction (PCR) was performed. IRAP-PCR analyses demonstrated that there were 0-70% polymorphism rates for HERV-K6, and 0-38% polymorphism rates for HERV-K11 among all the samples. Furthermore, the polymorphism rates were 0-70% among females and 11-60% among males for HERV-K6, and 0-38% among females and 0-25% among males for HERV-K11. Age-associated polymorphism was also investigated, but no age-associated polymorphism was observed among the samples. Therefore, HERV-K6 and HERV-K11 polymorphisms may arise on an individual-specific basis. Various previous studies have investigated the associations between the expression of HERVs and cancer or other major diseases. However, few reports have analysed HERV-K movements among individuals. This is the first report to investigate HERV-K6 and HERV-K11 retrotransposon polymorphisms between the genders and different age groups.

Current status and future possibilities of molecular genetics techniques in Brassica napus.

As PCR methods have improved over the last 15years, there has been an upsurge in the number of new DNA marker tools, which has allowed the generation of high-density molecular maps for all the key Brassica crop types. Biotechnology and molecular plant breeding have emerged as a significant tool for molecular understanding that led to a significant crop improvement in the Brassica napus species. Brassica napus possess a very complicated polyploidy-based genomics. The quantitative trait locus (QTL) is not sufficient to develop effective markers for trait introgression. In the coming years, the molecular marker techniques will be more effective to determine the whole genome impairing desired traits. Available genetic markers using the single-nucleotide sequence (SNP) technique and high-throughput sequencing are effective in determining the maps and genome polymorphisms amongst candidate genes and allele interactions. High-throughput sequencing and gene mapping techniques are involved in discovering new alleles and gene pairs, serving as a bridge between the gene map and genome evaluation. The decreasing cost for DNA sequencing will help in discovering full genome sequences with less resources and time. This review describes (1) the current use of integrated approaches, such as molecular marker technologies, to determine genome arrangements and interspecific outcomes combined with cost-effective genomes to increase the efficiency in prognostic breeding efforts. (2) It also focused on functional genomics, proteomics and field-based breeding practices to achieve insight into the genetics underlying both simple and complex traits in canola.

An Evaluation Model of Enhancing and Releasing Effect of the Blue Swimming Crab, Portunus trituberculatus, Based on Molecular Marker Technique

An Evaluation Model of Enhancing and Releasing Effect of the Blue Swimming Crab, Portunus trituberculatus, Based on Molecular Marker Technique

Identification of Three Egyptian Annona Cultivars Morhologically and Biochimically using Rapd Analysis

To detect remarkable fingerprinting and evaluate genetic variation and similarity among three Egyptian cultivarsAnnona sp. (which cultured in Sabahia Horticulture Research Station, during 2016 and 2017 seasons atSabahiaHorticulture Research Station (HRI), namely: Abd Elrazek, Baladi, and Senigalinsis) first morphologically using Leafdiameter, then seed number and fruit dimension, it was found that fruit and seeds could be used to evaluate variation andgenetic similarity among cultivars. Where Abdelrazik was the lowest in seed number with 37 seeds comparing with Baladiand Sengalinsis with 73 and 127 respectively.Moreover, fruit could be used to evaluate genetic similarity among cultivars. Abdelrzak was the lowest in seednumber with 37 seeds comparing with Baladi and Senigalinses with 73 and 127 respectively.Leaf parameters were used as a species and varieties diversity and found useful with longest leaf length forAbdelrzak and Sengalinsis 13.5 cm and 12.43 cm respectively. While Baladi was the shortest with 0.30 cm.`Finally molecular marker techniques (Random Amplified polymorphic DNA (RAPD) fingerprinting technique,)were used Different polymorphism percentages were recorded for four random primers (OPG1, POG2, OPG3 andOPG4) through this investigation. Generally, all genotypes could be distinguished via four random primers understudy. First primer reflected high polymorphic percentage with 82 % of polymorphism. On the other hand,high similarity percentage and lowest polymorphism percentage were recorded for fourth primer with 50 % ofpolymorphism.,,, A dendrogram was done to depict the pattern of relationships between the studied cultivars andtheir genetic diversity Not only it was fairly good But Also it reveal the need of further studies with new suitabletechniques.

Molecular Markers in Whole Genome Evolution of Brachypodium.

Molecular markers play more and more important role in population genetic and phylogenetic studies; choice of marker systems for a particular study has become a serious problem. These marker systems have different advantages and disadvantages, so it is imperative to keep in mind all the pros and cons of the technique while selecting one for the problem to be addressed.Here, we concisely introduced three molecular marker techniques, namely SSR, ISSR, and RFLP. We elaborated their properties such as reliability, simplicity, cost-effectiveness, and speed, in addition to data analysis of genetic diversity. We have outlined here the whole methodology of these techniques.

In vitro Propagation and Assessment of Genetic Relationships of Citrus Rootstocks Using ISSR Molecular Markers

The behavior of six citrus rootstocks, Volkameriana, Citrumelo ‘Swingle’, Citrange ‘Carrizo’, Poncirus trifoliata ‘Serra’, Poncirus trifoliata ‘Rubidoux’ and Poncirus trifoliata ‘Flying Dragon’, in in vitro propagation was studied and compared for shoot proliferation and rooting. In addition, the genetic relationships among the rootstocks studied and other Citrus species, using the Inter-Simple Sequence Repeats (ISSR) molecular markers, were investigated. Nodal explants of three months old shoots were used in Murashige and Skoog medium supplemented with N6-benzyladenine (BA) for shoot proliferation and with naphthaleneacetic acid (NAA) for rooting. The rootstock Volkameriana showed a statistically significant higher number of shoots (1.81), shoot length (15.14 mm) and number of leaves per explant (5.81), while all three Poncirus trifoliata rootstocks showed the lowest numbers. The number of roots and root length per explant were evaluated at the end of the rooting phase. The rootstock ‘Swingle’ showed a higher number of roots per explant (4.2) followed by ‘Flying Dragon’ (3.93) and ‘Carrizo’ (3.23) rootstocks. The rootstocks ‘Swingle’ (140.8 mm), Volkameriana (148 mm) and ‘Flying Dragon’ (131.12 mm) had significantly higher root length per explant compared to ‘Carrizo’ (31 mm) and ‘Rubidoux’ (34.5 mm). The ISSR molecular marker technique used in the present study grouped successfully the different species, varieties and rootstocks studied, revealing their genetic variability. The genetic variability observed among the rootstocks ranged between 0.29 (Poncirus trifoliata ‘Serra’ and Citrumelo ‘Swingle’) and 0.60 (Volkameriana and Citrumelo ‘Swingle’). The response of the rootstocks studied in in vitro propagation however is not related to their genetic affinity.

Dealing with AFLP genotyping errors to reveal genetic structure in Plukenetia volubilis (Euphorbiaceae) in the Peruvian Amazon.

An analysis of the population structure and genetic diversity for any organism often depends on one or more molecular marker techniques. Nonetheless, these techniques are not absolutely reliable because of various sources of errors arising during the genotyping process. Thus, a complex analysis of genotyping error was carried out with the AFLP method in 169 samples of the oil seed plant Plukenetia volubilis L. from small isolated subpopulations in the Peruvian Amazon. Samples were collected in nine localities from the region of San Martin. Analysis was done in eight datasets with a genotyping error from 0 to 5%. Using eleven primer combinations, 102 to 275 markers were obtained according to the dataset. It was found that it is only possible to obtain the most reliable and robust results through a multiple-level filtering process. Genotyping error and software set up influence both the estimation of population structure and genetic diversity, where in our case population number (K) varied between 2–9 depending on the dataset and statistical method used. Surprisingly, discrepancies in K number were caused more by statistical approaches than by genotyping errors themselves. However, for estimation of genetic diversity, the degree of genotyping error was critical because descriptive parameters (He, FST, PLP 5%) varied substantially (by at least 25%). Due to low gene flow, P. volubilis mostly consists of small isolated subpopulations (ΦPT = 0.252–0.323) with some degree of admixture given by socio-economic connectivity among the sites; a direct link between the genetic and geographic distances was not confirmed. The study illustrates the successful application of AFLP to infer genetic structure in non-model plants.

Phenotypic Diversities of Four Populations of Clarias Gariepinus (Siluriformes, Clariidae) Obtained from Ogun and Ondo State Waterbodies in South-Western Nigeria

Abstract The study determined the variation in the morphological and meristic features among four populations of Clarias gariepinus Burchell, 1822 obtained from Owena Dam and River Oluwa in Ondo State and Rivers Omo and Ogbere in Ogun State, both in Nigeria. A total of ninety five (95) and one hundred and twenty (120) fish specimens collected from Ondo and Ogun states respectively were measured using standard procedures and the results were analysed using Analysis of variance and multivariate analyses. The results obtained from the ANOVA and Principal Component Analyses of Clarias gariepinus from the four populations revealed heterogeneity for most of their characters. Therefore, the morphological differences between the wild African catfish found in Ondo and Ogun state populations could be linked to genetic differences or environmental factors or a combination of both factors. Hence, this study concluded that the populations are different which could imply high genetic diversity if molecular marker techniques are employed in further studies.

SHORT REVIEW ON DNA-FINGERPRINTING AND STANDARDIZATION OF HERBAL DRUGS

Medicinal plants play a key role in world health. In spite of new advances developed in modern medicines, herbal formulations are extensively used as therapeutic agents for treatment of several diseases. Herbal drug technology is used for converting botanicals materials into medicines. Standardization is a process of evaluating the quality & purity of crude drug by various parameters like morphological, microscopical, physical, chemical &biological observation. Fingerprint analysis approach is the most important in quality control of herbal medicines because of its accuracy and reliability. DNA fingerprinting is defined as the application of genetic/ molecular marker techniques which is used to identify cultivars. Fingerprinting is a process as it determines the concentrations of chemical substances in an herb by several methods like RFLP, AFLP, RAPD, SSR sequencing of rDNA-ITS region and DNA bar-coding. Development of fingerprints based on DNA markers is necessary for proper identification and standardization of plant species.

Sukkula retrotransposon movements in the human genome

ABSTRACTRetrotransposons, a subclass of mobile genetic elements, have been discovered in many organisms. In this study, we identified a plant-specific retrotransposon (Sukkula) in the human genome (24 different DNA samples) by using retrotransposon-based molecular marker technique: inter-retrotransposon amplified polymorphism. There were five different groups related to the ages of the subjects. The polymorphism ratios were 8%–100% among all samples, 10%–91% among females (12 subjects) and 13%–100% among males (12 subjects). Moreover, we also observed 8%–91% polymorphism ratios when comparing males to females. To the best of our knowledge, this is one of the first reports on plant-specific retrotransposons in the human genome. The obtained findings are expected to contribute to broadening the knowledge about plant retrotransposons in the human genome and their role in human genome evolution.

English

In this study, the genetic diversity and structure of 90 monosporidia mating-type isolates of Sporisorium scitamineum collected from China were comprehensively evaluated based on the DNA extracted from mating-type haploid sporidias using inter-simple sequence repeat (ISSR) polymorphism molecular marker technique. Fifteen selected ISSR primers produced 162 amplified DNA fragments, of which 115 (71.0%) were polymorphic. Polymorphic information content (PIC) values ranged from 0.12 to 0.92 with an average of 0.82, indicating that a moderate degree of genetic diversity exists among these 90 monosporidia mating-type isolates of S. scitamineum collected from China. Genetic differentiation coefficient (Gst) was calculated to be 0.320, indicating that the total genetic variation between populations was 32%, while 68% of the total genetic variation was from within populations. Meanwhile, gene flow (Nm) was 1.063, indicating lower rate of gene flow between populations. Based on the ISSR marker data set, the results of clustering by the unweighted pair group method of arithmetic average and principal component analysis were similar and could divide these 90 S. scitamineum isolates into two main groups. The isolates collected from the same province tend to cluster in the same main groups. This study shows that the genetic differentiation of these 90 monosporidia mating-type isolates of S. scitamineum was highly correlated with the ecological environment of collection sites, and the heterogeneity of ecological environment was the main driving force for the differentiation of S. scitamineum.   Key words: Sugarcane, Sporisorium scitamineum, mating-type haploid sporidia, inter-simple sequence repeat, genetic diversity.

Assessing Date Palm Genetic Diversity Using Different Molecular Markers.

Molecular marker technologies which rely on DNA analysis provide powerful tools to assess biodiversity at different levels, i.e., among and within species. A range of different molecular marker techniques have been developed and extensively applied for detecting variability in date palm at the DNA level. Recently, the employment of gene-targeting molecular marker approaches to study biodiversity and genetic variations in many plant species has increased the attention of researchers interested in date palm to carry out phylogenetic studies using these novel marker systems. Molecular markers are good indicators of genetic distances among accessions, because DNA-based markers are neutral in the face of selection. Here we describe the employment of multidisciplinary molecular marker approaches: amplified fragment length polymorphism (AFLP), start codon targeted (SCoT) polymorphism, conserved DNA-derived polymorphism (CDDP), intron-targeted amplified polymorphism (ITAP), simple sequence repeats (SSR), and random amplified polymorphic DNA (RAPD) to assess genetic diversity in date palm.

Evaluation of Clonal Fidelity of Micropropagated Date Palm by Random Amplified Polymorphic DNA (RAPD).

Date palm is a fruit-bearing tree commonly found in arid and semiarid regions. It is a dioecious plant, producing fruit on female plants and a limited number of basal offshoots for propagation. To produce large numbers of uniform plantlets, tissue culture techniques are required. It is highly advisable to detect genetic variation that may occur through micropropagation techniques as it may lead to phenotypic alterations. Random amplified polymorphic DNA (RAPD) is a simple and PCR-based molecular marker technique which can be employed to check the somaclonal variation. Screening of markers requires repeated confirmation of the pattern obtained in individual samples.

Review on Genetics and Breeding of Tomato (Lycopersicon esculentum Mill)

Tomato is considered as a member of the family Solanaceae. The botanical name of tomato is Lycopersicon esculentum Mill and is a diploid plant with 2n=2x=24 chromosomes. Great advances in tomato genetics have been achievable because of the understanding of mating systems and the possibility of controlled hybridization within and among species, the naturally occurring variability in the species, the occurrence of self-pollination that leads to the expression of recessive mutations, the lack of gene duplication, and the possibility to easily identify the 12 chromosomes. New methodological approaches like molecular mapping of important agronomical characters and the development of advanced-backcross and introgression lines have provided powerful tools for the improvement of the tomato crop and to understand the processes of domestication. The general breeding goals of tomato are fruit yield, fruit quality, and resistance to diseases and pests. Tomatoes are presented as a classical example for gene transfer from wild species into cultivated cultivars for improvement the qualitative traits. It was established that until now the achievements in tomato breeding are based on classical breeding-genetic methods and also, an essential change in the accelerated introduction of useful traits in the cultivars is not made. It is believable that conventional breeding wouldn’t allow the increase of productivity in the future. The significant progress in molecular genetics and use of molecular marker techniques are established. Therefore, the combined application of traditional breeding and contemporary plant biotechnology methods including selection based on molecular markers marker-Assisted Selection might be valuable tools for tomato breeding.

SSR analysis on stress effect of transgenic hybrid poplar 741 on Clostera anachoreta (Fabricius) (Lepidoptera: Notodontidae)

The genetic differentiation of the experimental population of Clostera anachoreta fed on different resistant transgenic 741 poplar leaves was analyzed by SSR molecular marker technique to investigate stress effect of transgenic poplar Bt gene as food on target insect. The experimental population of C. anachoreta fed on transgenic 741 poplar high resistant strains 'Pb29', medium resis-tant strains 'Pb17' and non-transgenic poplar (CK), and the screened ten pairs of SSR primers were used. The results showed that 76 alleles were observed in ten pairs of primers. The average allele was 7.6, the average effective number of alleles was 2.2, the average observed heterozygosity was 0.5167, the average expected heterozygosity was 0.5167, and the average percentage of polymorphic loci was 96.7%. The genetic diversity level of C. anachoreta experimental population fed on transgenic poplar 741 was significantly higher than that fed on non-transgenic populations, and C. anachoreta fed on high resistance had the lowest genetic similarity with CK samples, which showed an increasing trend of the genetic diversity of the experimental population fed on transgenic Bt poplar. It was thus clear that transgenic hybrid poplar 741 had stress effects on genetic differentiation of C. anachoreta experimental population by SSR.

Multiplex-ready PCR assay of SSR marker diversity among quality protein maize inbred parental lines

Phenotypic markers have long been used for classification of maize (Zea mays) cultivars. However, because of shortcomings in detecting differences among closely related genotypes, coupled with sensitivity to environmental conditions, molecular marker techniques have been employed in the determination of relationships among parental genotypes at the DNA level. In this study, simple sequence repeat (SSR) multiplex-ready PCR was used to determine the diversity among 24 quality protein maize (QPM) inbred lines. Analysis of SSR loci of the 24 QPM lines yielded 101 scorable alleles with an average of 5.05 alleles per locus. The highest polymorphic information content (0.77) was observed in di-repeat motifs (Bng 11714) and the least (0.15) in tetra-nucleotide repeat motifs (Phi 053). The expected and observed mean heterozygosities were 0.62 and 0.11, respectively. These values showed deviation from the Hardy–Weinberg equilibrium, an affirmation of genetic diversity among the maize accessions. UPGMA cluster analysis resolved four major clusters with grouping patterns consistent with ancestry information. The results indicate that the multiplex-ready PCR technique could avail breeders in developing countries, especially Africa where research funding is limited, the opportunity of making use of marker techniques in breeding programmes because it is highly economical and time efficient.