Application Of Molecular Markers Research Articles

Polymorphic Identification of Simple Sequence Repeat (SSR) Marker for Developing Aluminum-Tolerance Upland Rice

SSR marker is one of the genetic markers widely applied in plant breeding programs. The application of molecular markers in plant breeding is meant to accelerate the selection of cross-progeny. The research aimed to identify the SSR primers polymorphism between the parent and control that linked to Al tolerance and verify the cross-progeny of five crosses. The result gained from 37 SSR primers used in this study showed that only nine primers are polymorphic. These nine polymorphic primers are RM257, RM214, RM247, RM205, RM490, RM262, RM569, RM271, and RM19. The application of polymorphic markers on five cross-progeny which have shown the same band pattern as the parents and tolerant control on the use of 9 SSR primers recorded as follows: RM257 2 lines, RM214 5 lines, RM247 5 lines, RM205 lines, RM490 13 lines, RM262 5 lines, RM569 7 lines, RM271 4 lines, and RM19 6 lines. The selected SSR primers linked to Al tolerance in this research can be used as a reference for molecular breeding strategies to develop new Al tolerance rice varieties in dryland conditions.

The application of DNA molecular markers in the study of Codonopsis species genetic variation, a review

Codonopsis genus is comprised of species that are perennial plants primarily distributed across all east, southeast, and Central Asia. The most famous species of Codonopsis are C. tangshen, C. lanceolate, and C. pilosula. The records showed that they have a long story usage as traditional Chinese medicines, as they were alleged to be able to intensify the spleen and the lung as well as enriching blood and engendering liquid. Certain species have a culinary value in southern China and Southeast Asia, where they are considered as tea, wine, soup, plaster, and porridge. Codonopsis species were shown to be of great importance in medicine, due to their broad biological activity. Therefore, a clear understanding of their genetic diversity is needed. Adequate distinctions and descriptions of those species are necessary to preserve plant reservoir, investigations of genes associated with desirable traits, and understanding of evolutionary relationships. Subsequently, various molecular marker techniques such as Random Amplified Polymorphic DNA (RAPD), Amplified Fragment Length Polymorphism (AFLP), Simple Sequence Repeats (SSR), and Inter Simple Sequence Repeat (ISSR), Single Nucleotide Polymorphism (SNP), internal transcribed spacer (ITS), and Sequence-Characterized Amplified Region (SCAR) have been improved to provide detailed informations about genomes, that historically were not possible to obtain based on only phenotypic methods. This review represents the usage of DNA molecular markers for molecular diversity analysis of medically important species belonging to the genus Codonopsis.

Identification and Validation of an Immune-Related Gene-Based Prognostic Index for Bladder Cancer

Background: Bladder cancer (BLCA) is a common malignant tumour of the urinary system. Knowledge of the mechanism of and treatment methods for BLCA remain limited. Methods: Robust rank aggregation was used to integrate five GEO bladder cancer microarray datasets for identifying differentially expressed genes (DEGs) between NMIBC and MIBC. An immune-related gene (IRG)-based prognostic index (IRGPI) was constructed using multivariate analysis. The reliability of the IRGPI was evaluated using independent datasets, and correlations between the IRGPI and clinicopathological characteristics as well as the immune microenvironment were evaluated. Finally, a nomogram was established to evaluate patient prognosis with BLCA. Findings: We identified 154 DEGs related to the degree of BLCA infiltration, including 24 IRGs. Missense mutation was the most common mutation type in IRGs. Ten hub IRGs were identified by protein-protein interaction network analysis. GSEA and GSVA of two novel BLCA-related genes (TYROBP and FCER1G) revealed that they were related to immunity. Nine survival-related IRGs were identified and their potential regulation by transcription factors was analysed. An independent IRGPI (CTSE, CXCL10, FAM3B, MMP9, OLR1, and S100P) was established, and was confirmed to be related to tumour stage, tumour grade, T stage, and N stage. The nomogram could effectively predict the prognosis of BLCA patients. Importantly, the IRGPI reflected infiltration by several types of immune cells. Interpretation: Our data provide new insights in the pathogenesis of BLCA and target genes for immunotherapy. The application of molecular markers for hierarchical prediction paves the way to precision medicine. Funding: This study was financially supported by the Chinese Academy of Medical Sciences Innovation Fund for Medical Sciences (grant number: 2018-I2M-1-002) and the Beijing Hospital Clinical Research 121 Project (BJ-2018-090). Declaration of Interest: The authors have declared no competing interests.

Development and application of chloroplast molecular markers in Panax notoginseng

The molecular markers(cpSSR, cpSNP and cpIndel) were developed based on the whole genome sequence of Panax notoginseng chloroplast genome, which provide a powerful tool for the evaluation and analysis of the future P. notoginseng germplasm resources. The 89 P. notoginseng samples from 9 groups were used for the experiment, and the data for the study were derived from NCBI and the GenBank numbers were: KJ566590, KP036468, KR021381 and KT001509. Through sequence alignment, 30 polymorphic sites(SNP and Indel) were identified, including 16 cpSNP and 14 cpIndel; cpSNP and cpIndel accounted for far more than the gene region in the intergenic region. The developed cpSSR reached 87-89, the repeat unit was mainly composed of trinucleotide, accounting for 70%-71%, and the dinucleotide was the least, accounting for 7%. Eighteen cpDNA molecular markers were developed, including 7 cpSSR primers, 6 cpIndel primers, and 5 cpSNP primers. The MatK gene and ycf1 primers were chosen as control. According to the results of DNA gel electrophoresis, cpSSR-5, pgcpir019 and pncp08 can be used to distinguish different cultivated populations of P. notoginseng. Among them, cpSSR-5 and pgcpir019 can also be used to distinguish the inter-species resources of ginseng by comprehensive sequence length, population π value and average nucleotide difference. However, pncp08 can only be used to distinguish different populations of P. notoginseng. In addition, the effect of distinguishing the groups of P. notoginseng, which the primer pncp-M(based on the MatK gene) is weaker than the cpSSR-5, pgcpir019 and pncp08.

Genetic and Phenotypic Characterization of Rice Backcrossed Inbred Sister Lines of Saltol in Temperate Saline Reclaimed Area

Saltol is one of the most well-known quantitative loci (QTLs) for salinity tolerance in rice. It has been used to develop highly tolerant rice varieties in saline and coastal areas in Southeast Asia, South Asia, and Africa. However, the functional activity of Saltol is not well known, and the molecular marker application of readily developed linked markers in Saltol has not always been successful in the rice breeding programs for salinity tolerance improvement. Interestingly, two BC2F9 sister backcrossed inbred lines (BILs), which have been developed by marker-assisted backcrossing utilized the linked markers of Saltol to improve the salinity tolerance of MS11 (a temperate japonica growing in tropical condition). The BILs showed very different phenotypic and stress tolerance, although both contained the Saltol QTL. The genomic similarity of the two BILs was 73%, and we have identified the genomic sites of different genic constitutions between the lines utilizing background genotyping. The stress response of the two BILs showed difference in survival rate, grain yield under highly saline field condition, and SPAD, SES in hydroponic conditions. MS11-SaltolA showed salinity tolerance through Na+/K+ homeostasis with relatively high K+ ion uptake and low Na+ ion uptake in the seedling stage. Further genomic analyses with whole genome resequencing is ongoing to study on gene interactions. The developed highly tolerant MS11-SaltolA can be used as an improved donor in rice molecular breeding for high salinity tolerance.

Studies on heritability and genetic variability for grain physical properties in Malaysian rice germplasm

Genetic based information of different traits plays important role in varietal improvement of rice. Twenty rice accessions (Oryza sativa L.) obtained from National Rice Gene Bank were evaluated during main season 2015/2016 to estimate heritability, genetic variability and genetic advance for seven grain physical properties. Among the traits, milled grain length/width ratio, milled grain length, milled grain length after cooking and grain length exhibited high estimates of phenotypic coefficient of variation (PCV), and genotypic coefficient of variation (GCV). Highest broad sense heritability and genetic advance was obtained for grain length (98.66% and 62.25%), milled grain length (98.95% and 67.31%), milled grain length/width ratio (98.50% and 80.41%) and milled grain length after cooking (98.93% and 65.44%) which suggest these traits are most probably controlled by additive gene action and hence they can be fixed by selection. However, kernel elongation ratio showed lowest value of broad sense heritability and genetic advance (11.76% and 1.38%, respectively) and may suggest non-additive gene action in their inheritance and selection of this trait may difficult due to high environmental influences. Therefore, improvement of high quality rice with kernel elongation ability may require molecular marker application as it is highly affected by environment for precise selection.

Molecular markers in the genetic diversity studies of representatives of the genus Rubus L. and prospects of their application in breeding

Род Rubus L. (семейство Rosaceae Juss.), по оценкам разных систематиков, состоит из 12–16 подродов, объединяющих ~750 видов. Самые крупные по числу видов подроды – Idaeobatus (Focke) Focke, к которому относятся малины, и типовой подрод Rubus (=Eubatus Focke), включающий виды ежевик. Представители рода Rubus обладают высокой пищевой и хозяйственной ценностью, а также лекарственными свойствами. Селекционные исследования направлены на расширение генетического разнообразия и создание новых сортов малин и ежевик, устойчивых к биотическим и абиотическим стрессорам и отличающихся высоким качеством плодов. Современные селекционно-генетические программы все шире включают достижения молекулярной генетики и геномики. В данной статье представлен обзор фундаментальных и прикладных исследований генетического разнообразия культивируемых и дикорастущих видов рода Rubus, выполненных на основе методов молекулярного маркирования. Рассмотрены основные типы молекулярных маркеров (RFLP, RAPD, SSR, ISSR, AFLP, SCAR, SSCP, ретротранспозонные маркеры и т. д.) и области их применения в изучении представителей рода Rubus. Приведены результаты работ по использованию методов ДНК-маркирования для решения самых разных задач, включая: исследование межвидового и внутривидового генетического разнообразия, филогенетических связей видов и надвидовых таксонов, выяснение спорных вопросов систематики, генотипирование и уточнение родословных сортов малин и ежевик, изучение сомаклональной изменчивости и др. Наиболее важным результатом в практическом плане является создание насыщенных молекулярно-генетических карт для разных видов малин и ежевик, на которых локализованы многочисленные гены и QTL, детерминирующие различные хозяйственно ценные признаки. В то же время необходимо отметить, что число маркеров, перспективных для проведения эффективного молекулярного скрининга, пока еще недостаточно.

Phylogenetic Position of Pleurogenoides Species (Plagiorchiida: Pleurogenidae) from the Duodenum of Indian Skipper Frog, Euphlyctis Cyanophlyctis (Amphibia: Dicroglossidae) Inhabiting the Western Ghats, India.

SummaryTwo species of digenetic trematodes of the genus Pleurogenoides viz., P. cyanophlycti Shinad & Prasadan (2018a) and P. euphlycti Shinad & Prasadan (2018b) have been described from India. Information regarding the molecular data of various species of the genus Pleurogenoides Travassos, 1921 is virtually lacking. This study addresses the application of molecular markers to validate the phylogenetic position of P. cyanophlycti and P. euphlycti. In the present study, two species P. cyanophlycti and P. euphlycti were collected between January 2016 to October 2017, infecting the freshwater frogs inhabiting the Western Ghats, India. In the present study, the two species were identifi ed morphologically and by PCR amplification of the 28S ribosomal RNA gene. Phylogenetic tree results clearly demonstrate that both P. cyanophlycti and P. euphlycti belongs to the family Pleurogenidae Looss, 1899. Based on these results, we presented and discussed the phylogenetic relationships of P. cyanophlycti and P. euphlycti within family Pleurogenidae from India. Phylogenetic analyses showed that P. cyanophlycti and P. euphlycti cluster according to their vertebrate host and revealed an important congruence between the phylogenetic trees of Pleurogenoides and of their vertebrate hosts. P. cyanophlycti and P. euphlycti clearly constitute a separate, sister branch with other species of the genera, Pleurogenoides, Pleurogenes (=Candidotrema), Prosotocus and Brandesia. The present study firstly provides important information about the molecular study and phylogenetic analysis of P. cyanophlycti and P. euphlycti. This study will also serve as a baseline for Pleurogenoides species identifi cation for further studies.

The Application of UTY and SRY Molecular Markers for Determination of Unknown Sex Samples in Bali Cattle

An investigation involving large number of animals is often resulting incomplete or in accurate information such as animal parentage, or misidentify on sex due to unlabeled sex samples. A PCR method by applying Y chromosome markers (UTY and SRY) facilitates in determination of unknown sex problem. This study was intended to determine sex from unlabelled sex of blood samples by applying PCR method using a pooled-DNA template. Twenty five of unknown sex blood samples from Nusa Penida, Bali were used in this study. The samples were plotted into 5 pooled-DNA whith each pool DNA consisted of 5 individuals DNA. Two pairs of sex primers, UTY (58oC) and SRY (60oC) with 35 cycles were applied to amplify the samples. The result showed there was only one pooled-DNA (P4) amplified by UTY (484bp). Whereas re-PCR of the positive pooled-DNA (P4) using SRY primer, only one out of 25 samples determined as male Bali cattle (325bp). This finding suggests that UTY and SRY primers are suitable for sex determination and the pooled-DNA could be used as an efficient PCR method both in consumables and PCR process for sex determination. Keywords: Determination, sex, unknown sample, pooled DNA, Bali cattle.

Applications of molecular markers in conserving endangered species

Applications of molecular markers in conserving endangered species

Fiber Quality Improvement in Upland Cotton (Gossypium hirsutum L.): Quantitative Trait Loci Mapping and Marker Assisted Selection Application.

Genetic improvement in fiber quality is one of the main challenges for cotton breeders. Fiber quality traits are controlled by multiple genes and are classified as complex quantitative traits, with a negative relationship with yield potential, so the genetic gain is low in traditional genetic improvement by phenotypic selection. The availability of Gossypium genomic sequences facilitates the development of high-throughput molecular markers, quantitative trait loci (QTL) fine mapping and gene identification, which helps us to validate candidate genes and to use marker assisted selection (MAS) on fiber quality in breeding programs. Based on developments of high density linkage maps, QTLs fine mapping, marker selection and omics, we have performed trait dissection on fiber quality traits in diverse populations of upland cotton. QTL mapping combined with multi-omics approaches such as, RNA sequencing datasets to identify differentially expressed genes have benefited the improvement of fiber quality. In this review, we discuss the application of molecular markers, QTL mapping and MAS for fiber quality improvement in upland cotton.

Advances in molecular biology of Paeonia L.

Molecular biology can serve as a tool to solve the limitations of traditional breeding and cultivation techniques related to flower patterns, the improvement of flower color, and the regulation of flowering and stress resistance. These characteristics of molecular biology ensured its significant role in improving the efficiency of breeding and germplasm amelioration of Paeonia. This review describes the advances in molecular biology of Paeonia, including: (1) the application of molecular markers; (2) genomics, transcriptomics, proteomics, metabolomics, and microRNA studies; (3) studies of functional genes; and (4) molecular biology techniques. This review also points out select limitations in current molecular biology, analyzes the direction of Paeonia molecular biology research, and provides advice for future research objectives.

New Male Specific Markers for Hop and Application in Breeding Program

Male specific DNA sequences were selected from a Diversity Arrays Technology (DArT) mapping study to evaluate their suitability for determination of the sex phenotype among young seedlings in a hop (Humulus lupulus L.) breeding program. Ten male specific DArT markers showed complete linkage with male sex phenotype in three crossing families. Following optimization, four were successfully converted into PCR markers and a multiplex PCR approach for their use was developed. Among 197 plants (97 from the world collection; 100 from three segregating families), 94–100% positive correlation with sex phenotypic data was achieved for the single PCR amplification, whereas the multiplex approach showed 100% correlation. To develop a fast and low-cost method, crude sample multiplex PCR was evaluated in 253 progenies from 14 segregating populations without losing accuracy. The study describes, for the first time, the routine application of molecular markers linked to male sex in an intensive Slovenian hop breeding program. The methods described could be employed for screening of sex at the seedling stage in other hop programs worldwide, thereby saving resources for desirable female plants.

Application of Molecular Markers and their Utility in Genetic Studies of Floricultural Crops: A Review

Understanding the genetics of floricultural germplasm represents a value added component of managing collections. Molecular approaches collectively represent a potential goldmine of important information that can be applied to programmes of genetic improvement. The development and utilization of molecular markers for the exploitation and identification of plant genetic diversity is one of the most key developments in the field of molecular genetics studies. Molecular markers are popular tool, due to their stability, cost-effectiveness and ease of use for a variety of applications in the field of molecular genetics. To know the role of molecular markers in genetic studies of floricultural crops, a number of molecular marker techniques have been reviewed by many researchers in worldwide. However, all these reviews were meant for researchers with advanced knowledge of molecular markers and their utility in limited aspects under floricultural crops. This review presents an overview of different marker technologies and their variants with a comparative account of their characteristic features and applications especially diverse molecular techniques used in assessing plant genetic diversity, inter-relationship between the genotypes, genetic purity, DNA barcode, tagging of important genes, development of linkage maps, marker assisted selection and application of molecular markers in tissue culture of floricultural crops. Altogether, the characteristics and types of markers with their applications and some recent advancement in genetic studies of floricultural crops are discussed.

Application of DNA Molecular Markers in Plant Breeding (Review article)

Application of DNA Molecular Markers in Plant Breeding (Review article)

Creation of an avocado unambiguous genotype SNP database for germplasm curation and as an aid to breeders

Avocado (Persea americana) is an important tropical and subtropical fruit tree crop. Traditional tree breeding programs face the challenges of long generation times and significant expense in land and personnel resources. Avocado selection and breeding can be more efficient and less expensive through the development and application of molecular markers. A total of 1524 individuals were genotyped with 384 SNPs creating the largest SNP genotype database for avocado. These individuals correspond to four extensive germplasm collections including two housed in Florida and two in California. In addition, hybrids and selections from two rootstock breeding programs have been genotyped. Genotype data were analyzed using an affinity propagation method to define 155 groups. The 384 SNP markers provided accurate genotype data for individuals from different Persea species as well as half-siblings. Therefore, the majority of the genetic diversity of the avocado germplasm and related species that were genotyped has been captured. A simple visual method can also be used to identify self-pollinated individuals among the half-siblings of known maternal parents and, in some cases, to infer likely candidates for the paternal parent. Finally, this dataset is unambiguous so breeders can determine the genetic diversity of their breeding stock to optimize avocado breeding and selection programs by identifying outcrossed individuals at the seedling stage, thus increasing the efficiency of avocado genetic improvement.

Advances in the diagnostic pathological features of precancerous lesions of hepatocellular carcinoma

The occurrence of hepatocellular carcinoma (HCC) is a multistep development process through precancerous lesions. A precancerous lesion of HCC is classified into hepatocyte dysplasia at the cytological level and dysplastic nodules at the histological level, and the corresponding lesion subtypes have different risks of canceration. Pathology is the "gold standard" for the diagnosis of early stage HCC and its precancerous lesions. However, it also faces many difficulties and challenges, such as the accumulation of experience in the pathological diagnosis, the understanding and grasp of key points of histopathological diagnosis and differential diagnosis, the combination application of immune and molecular diagnostic markers, and many others. This article briefly discusses the key points of pathological features and differential diagnosis of precancerous lesions of HCC.

Clinical significance of the histological and molecular characteristics of ependymal tumors: a single institution case series from China

BackgroundEpendymal tumors are pathologically defined intrinsic neoplasms originating in the intracranial compartments or the spinal cord that affect both children and adults. The recently integrated classification of ependymomas based on both histological and molecular characteristics is capable of subgrouping patients with various prognoses. However, the application of histological and molecular markers in Chinese patients with ependymomas has rarely been reported. We aimed to demonstrate the significance of histological characteristics, the v-relavian reticuloendotheliosis viral oncogene homolog A (RELA) fusions and other molecular features in ependymal tumors.MethodsWe reviewed the histological characteristics of ependymal tumors using conventional pathological slides and investigate the RELA fusions and Cylclin D1 (CCND1) amplification by Fluorescence in situ hybridization (FISH) and trimethylation of histone 3 lysine 27 (H3K27me3) expression by immunohistochemistry (IHC) methods. SPSS software was used to analyze the data.ResultsWe demonstrated that hypercellularity, atypia, microvascular proliferation, necrosis, mitosis, and an elevated Ki-67 index, were tightly associated with an advanced tumor grade. Tumor location, necrosis, mitosis and the Ki-67 index were related to the survival of the ependymomas, but Ki67 was the only independent prognostic factor. Additionally, RELA fusions, mostly presented in pediatric grade III intracranial ependymomas, indicated decreased survival times of patients, and closely related to the patients’ age, tumor grade, cellularity, cellular atypia, necrosis and Ki67 index in the intracranial ependymal tumors, whereas reduction of H3K27me3 predicted the worse prognosis in ependymal tumors.ConclusionsHistological and molecular features facilitate tumor grading and prognostic predictions for ependymal tumors in Chinese patients.

Identification of driver genes and somatic mutations in cell-free DNA of patients with pulmonary lymphangioleiomyomatosis.

Next-generation sequencing of cell-free circulating DNA (cfDNA) has emerged as promising technique for identifying minimally invasive genomic profiling of tumor cells recently. However, it remains relatively unknown in LAM disease. In our study, paired cfDNA and genomic DNA (gDNA) in blood samples were obtained from 23 LAM patients and seven healthy controls to explore mutations profiles of targeted 70 cancer-related genes. As results, log2-based allele frequencies of mutations in cfDNA were significantly different from those of gDNA. By comparing the mutual mutations identified both in cfDNA and gDNA, a significant correlation was also observed. After removing mutations in gDNA, distinct somatic mutation profiles of cfDNA were observed in LAM patients. Forty of 70 targeted genes had recurrent mutations, of which ATM, BRCA2 and APC showed the highest frequency. Based on the mutation, correlation network constructed of 40 mutated genes, 11 hub genes bearing intensive interactions were highlighted, including BRCA1, BRCA2, RAD50, RB1, NF1, APC, MLH3, ATM, PDGFRA, PALB2 and BLM. Expression of the hub genes showed significant clusters between LAM patients and controls and that RAD50 and BRCA2 had the strongest associations with subject phenotypes. Myogenesis and estrogen response were confirmed to be positively regulated in LAM patients. Collectively, our study provided a landscape of genomic alterations in LAM and discovered several potential driver genes, that is, BRCA2 and RAD50, which shed a substantial light on the clinical application of key molecular markers and potential therapy targets for precision diagnosis and treatment in the future.

THE RELATIONSHIP BETWEEN RAPD MARKER-BY-MARKER INTERACTIONS AND QUANTITATIVE TRAITS OF CARAWAY (Carum carvi L.)

Application of molecular markers makes the selection process much more effective. Marker assisted selection is an important tool for plant breeders to increase the efficiency of a breeding process, especially for multigenic traits, highly influenced by the environment. Epistasis is the interaction between alleles from two or more loci determining the complex traits, and thus plays an important role in the development of quantitative traits of crops. In this paper, the relationships between RAPD marker-by-marker interactions and 22 quantitative traits of caraway (Carum carvi L.) were analyzed. Significant associations of 116 epistatic markers with at least one trait in 2004 as well as 112 in 2005 were found on the basis of multivariate regression analysis. The proportion of total phenotypic variances of individual trait explained by the marker-by-marker interactions ranged from 25.3% to 96.0%.